Talk:MiPNet17.08 IOC68 Schroecken AT

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IOC68 Workshop Experiments

2012-04-14 Demo Experiment

• Oxygraph AB (in both chambers the same SUIT protocol was applied)
• DatLab File 2012-04-14_AB-02
• mouse heart homogenate prepared with PBI Shredder (position 1 for 10 seconds, position 2 for 5 seconds) and the homogenate was completely washed out of the Shredder tube with 5 ml MiR05
• 2.3 mg tissue per chamber = 1.15 mg / ml
• Flux derivation = 20
• oxygen regime 320 to 160 (reoxygenation with pure oxygen at open chamber position)
• respiration medium: MiR05 (solubility factor = 0.92)
• SUIT-Protocol: PMG + AmR + HRP + H₂O₂ + D2 + D3 + S + FCCP + Rot + H₂O₂ + H₂O₂ + Mna + Ama
• the homogenate was added, the oxygen concentration was increased till 320 nmol/ml and then the chamber was closed

Notes

• following states were obtained:

CI-linked L respiration with 5 mM pyruvate, 2 mM malate and 10 mM glutamate in the absence of ADP (no adenylates, N)

ADP-stimulated state D after titration of 3 mM ADP

CI+II-linked respiration after titration of 10 mM succinate

uncoupling with 0.5 µM FCCP

S(Rot) with 0.5 µM rotenone.

• titration of 3 mM ADP stimulated the oxygen flux about 69.0 to 72.7 % and inhibited the peroxide production 1.0 to 4.7 %
• addition of succinate stimulated respiration in both chambers about 27 % whereas H₂O₂ flux was reduced about 2.6 to 9.2 %.
• reoxygenation with pure oxygen increased the O₂ flux (14.3 - 17.5 %) as well as the H₂O₂ flux (19.2 - 26.1 %)
• a direct influence of the O2 concentration on peroxide production as an induction of peroxide production occurred after reoxygenation with pure oxygen.
• FCCP reduced the oxygen flux (2.7 – 6.6 %) and the peroxide production (3.1 to 5 fold; fig. 9 A and B). In the end of the experiment, rotenone as inhibitor of CI was added, which reduced the oxygen flux (2 fold decrease) as well as the H2O2 flux (25 - 29 %) in both chambers.
• in both chambers, the highest H₂O₂ / O₂ flux ratio (L with PMG) was 0.0034 (0.34 %) and the addition of ADP diminished the H₂O₂ / O₂ flux ratio to 0.0010 (0.10 %)
• titration of succinate further diminished the ratio to 0.0007 (0.07 %) and 0.5 µM FCCP resulted in a H₂O₂ / O₂ flux ratio of 0.0002 (0.02 %)
• reoxygenation caused an increase to 0.0007 (0.07 %) and rotenone further increased the ratio to 0.0013 (0.13 %).
• oxygen dependence of homogenate became visible as a decline of the oxygen flux and the H₂O₂ flux together with the decline of the oxygen concentration occurred indicating a dependence of oxygen with homogenate that is not that strong as observed with fibres.
• analysis of the experiments is difficult because of O₂ dependence