4.9. High-Resolution Respirometry

Respiration was measured at 37 °C under constant stirring (750 rpm), which ensured a homogenous oxygen distribution in the medium in a high-resolution respirometer using an Oxygraph-2k (O2k, Oroboros Instruments, Innsbruck, Austria), a modular system for high-resolution respirometry (HRR) according to Volani et al. [89]. Oxygen concentration (μM) and oxygen flux per mass (pmol O₂● s⁻¹·mg⁻¹) were recorded in real-time, while obtained data were evaluated using DatLab software (Oroboros Instruments, Innsbruck, Austria). Briefly, mitochondrial respiration was measured in MiR05 (mitochondrial respiration medium containing 0.5 mM of EGTA, 3 mM of MgCl₂·6H₂O, 60 mM of potassium lactobionate, 20 mM of taurine, 10 mM of KH₂PO₄, 20 mM of HEPES, 110 mM of sucrose, and 1 g/L of BSA fatty acid-free (pH 7.1) (MiR05; Oroboros Instruments, Innsbruck, Austria). Freshly isolated liver mitochondria (0.1 mg of mitochondrial protein per each 2-mL chamber) used for respiration measurement were first incubated for 5 min in the medium without (control) or in presence of Mel, 6(OH)Mel, or 5-MT.

For the assessment of mitochondrial respiration, the following protocol was used:

Non-phosphorylating LEAK respiration was assessed by injecting 10 mM of sodium pyruvate, 10 mM of l-glutamic acid (neutralized with KOH) and 2 mM of l-malic acid (neutralized with KOH) as NADH (N)-linked substrates: state NL;

OXPHOS capacity was induced by adding 1.25 mM of ADP at saturating concentration: state NP;

10 μM of cytochrome c was added to test the integrity of the outer mitochondrial membrane: state NC;

NADH and succinate (NS)-linked OXPHOS capacity was measured by adding 10 mM of succinic acid (neutralized with KOH): state NSP.