Timon-Gomez 2023 GIBB
Event: GIBB 2023 Riva del Garda IT
Comparative OXPHOS analysis is essential for understanding the physiological implications of tissue- and species-specific respiratory coupling and pathway control patterns. Their analysis is an essential component of the diagnosis of mitochondrial diseases. To address these challenges, we developed a substrate-uncoupler-inhibitor titration reference protocol (SUIT-RP) for measuring metabolic fluxes by high-resolution respirometry. The scope of investigations into mitochondrial respiratory function is limited when using living cells, due to the impermeability of the plasma membrane to certain compounds and mitochondrial preparations are required for OXPHOS analysis.
The SUIT-RP protocol consists of two complementary protocols, RP1 and RP2, that evaluate 20 respiratory states with multiple electron entries feeding the Q-junction and with cross-linked states for harmonization. Defined coupling and pathway states are established in different mitochondrial preparations (isolated mitochondria, tissue homogenate, permeabilized cells). RP1 focuses on NADH-linked linear coupling control (LEAK-OXPHOS-ET), separating coupling control and substrate control. RP2 evaluates the effect of fatty acid oxidation (FAO) in the OXPHOS state. We demonstrated the applicability of SUIT-RP in different tissues (heart, brain, blood), and organisms (human and mouse models).
Importantly, the SUIT-RP protocol was designed to avoid classical artifacts in measuring FAO pathway and Complex IV activity, which have previously confounded research in the field. The SUIT-RP protocol represents a valuable baseline for developing a mitochondrial pathway and coupling control database, which can contribute to a better understanding of the diversity of mitochondrial function and to comparative studies for functional diagnosis in health and disease.
Labels: MiParea: Respiration
- Oroboros Instruments, Innsbruck, Austria