Template:SUIT-034 NADH mt D082

Steps and respiratory states

Step	State	Pathway	Q-junction	Comment - Events (E) and Marks (M)
mt	REN			mt Respiration in the REN state is due to the presence of residual endogenous substrates. In the absence of endogenous substrates, mt can be used for calibration of fully oxidized NAD.
1D.1				mt;1D.1 The addition of a low concentration of ADP (0.1 μM) stimulates the consumption of endogenous substrates, which can be observed as a decrease in respiration.
2PGM	PGM	N	CI	mt;1D.1;2PGM NADH-linked substrates (type N-pathway to Q). The addition of fuel substrates may trigger a transient peak increase in mitochondrial respiration due to the presence of low ADP concentrations. Once ADP is phosphorylated to ATP the respiration decreases. In the presence of ATPases in the sample, ATP is recycled to ADP, making it not possible to measure LEAK state.
3D2.5	PGM_P	N	CI	mt;1D.1;2PGM;3D2.5 NADH-linked substrates (type N-pathway to Q). OXPHOS capacity P (with saturating [ADP]), active OXPHOS state.
4U	PGM_E	N	CI	mt;1D.1;2PGM;3D2.5;4U NADH-linked substrates (type N-pathway to Q). Uncoupler titration (avoiding inhibition by high uncoupler concentrations) to obtain electron transfer (ET) capacity E (noncoupled ET-state). Test for limitation of OXPHOS capacity P by the phosphorylation system (ANT, ATP synthase, phosphate transporter) relative to ET capacity E in mt-preparations: E-P control efficiency and E-L coupling efficiency. In living cells: E-R control efficiency and E-L coupling efficiency.
5Anox		N	CI	mt;1D.1;2PGM;3D2.5;4U;5Anox Anoxia, after the biological sample has consumed the O₂ in the O2k-chamber, is used to detect the fully reduced NAD for calculation of the NAD redox states.
6Myx		N	CI	mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx The addition of myxothiazol after anoxia is a crucial step to detect the fully reduced NAD for calculation of the NAD redox state. Myxothiazol can induce a further reduction of NAD under anoxia.
7Reox	ROX			mt;1D.1;2PGM;3D2.5;4U;5Anox;6Myx;7Reox The reoxygenation by opening the chamber in the presence of Myx allows for Rox-correction of the O₂ fluxes.