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. 2022 May 10;11(10):1598. doi: 10.3390/cells11101598

Figure 2.

Figure 2

Assay chemistry and instrumentation. (A) The ATP:O ratio represents the ratio of ATP synthesis based on oxygen consumption measured simultaneously. It allows us to measure the efficiency of the oxidative phosphorylation. This ratio is determined using the “hexokinase system” based on the regeneration of ADP [17,25], thus making it possible to obtain a stable State 3. The intensity of State 3 can be modulated by increasing concentrations of ADP, resulting in the kinetics of ATP synthesis being obtained as a function of the consumption of oxygen between State 4 (the absence of ATP synthesis) and State 3 (maximum ATP synthesis). The reaction catalyzed by hexokinase (HK) is as follows: synthesized ATP is measured indirectly by evaluating glucose-6-phosphate production during the reaction. Glucose-6-phosphate dehydrogenase (G6PDH) allows the formation of NADPH from glucose-6-phosphate: HK glucose + ATP gGlucose-6-phosphate + ADP. The quantity of NADPH formed during this reaction can then be measured with a spectrophotometer at a wavelength of 340 nm and is directly proportional to the amount of ATP synthesized. (B) Typical trace of mitochondrial respiration. The respiration rate (determined by the slope of the curve) is calculated by the amount of consumed oxygen/minute/mg of protein. The State 2 non-phosphorylating state is respiration in the absence of ATP synthesis. State 3 is respiration in the presence of ATP synthesis. State 4 is the respiration in the presence of oligomycin, an ATP synthase inhibitor. Cytochrome c oxidase activity is the maximal respiration with ascorbate and N,N,N′N′-tetra methyl-p-phenylenediamine (TMPD) as a substrate. (C) ATP:O is the linear relationship between ATP production and oxygen consumption measured with the addition of different amounts of exogenous ADP. The mitochondrial ATP synthesis is initiated by the addition of different amounts of ADP. The intensity of State 3 can be modulated by increasing the concentration of ADP, resulting in the kinetics of ATP synthesis being obtained as a function of oxygen consumption between State 4 (absence of ATP synthesis) and State 3 (maximum ATP synthesis). There is a linear relationship between ATP synthesis and oxygen consumption when the oxidative phosphorylation rate increases by the addition of higher amounts of ADP.