Absolute Quantitation

Introduction



The Iberius cell spectroscopy system measures oxidation changes from baseline. It is necessary to obtain the fully reduced state and the fully oxidized state in order to calculate the oxidation state (percent of each heme oxidized) and content from the oxidation changes.

The fully reduced state is obtained by decreasing the oxygen concentration to zero (anoxia) for 2 minutes. This prevents electrons leaving the chain at cytochrome oxidase and they build up on the chain leading to full reduction of the hemes and mitochondrial NADH from which the fully reduced level can be determined (Figure 1).

The oxygen is then returned to 100μM and at this point compounds can be added to the cells to determine their effects.
At the end of the study, the cells are treated with a high dose of uncoupler (U) which collapsed ΔP and atpenin a5, which is a complex II inhibitor and also results in inhibits the TCA cycle. The combination of TCA cycle inhibition and collapse of ΔP results in the electron transport chain fully oxidizing the mitochondrial NADH pool to give the NADH fully oxidized state. The oxidation state of mitochondrial NADH can be back-calculated at all times.

Finally, the cells are treated with either rotenone or piericidin, which are complex I inhibitors. The combination of complex I and complex II inhibition leads to complete blockage of electrons entering the chain and full oxidation of all the hemes to give their fully oxidized state. The content of each heme, and hence the content of the complex which contains the heme, can be calculated from the difference between the fully oxidized and fully reduced state, and the oxidation state back calculated.

All these calculations are carried out automatically by the Iberius software on completion of a study.

Figure 1 from oxidation changes to absolute quantitation. In all cases, a downward deflection denotes reduction and an upward deflection denotes oxidation. Additions: Uncoupler (U), Atpenin a5 (A) and rotenone (R).
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