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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 +U(FCCP) +Rot +H2O2 +H2O2 +Mna +Ama
  • The homogenate was added, the oxygen concentration was increased up to 320 nmol/ml and then the O2k-chamber was closed.

Notes

  • The following respiratory 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 P after titration of 3 mM ADP.
CI&II-linked respiration after titration of 10 mM succinate.
Noncoupled state E at 0.5 ยตM FCCP.
S(Rot) (CII-linked) state E with 0.5 ยตM rotenone.
  • Titration of 3 mM ADP stimulated the oxygen flux about 69 to 73% and inhibited peroxide production by 1.0 to 4.7%.
  • Addition of succinate stimulated respiration in both chambers by about 27%, whereas H2O2 flux was reduced by about 2.6 to 9.2%.
  • Reoxygenation with pure oxygen increased the O2 flux (14 - 18%) as well as the H2O2 flux (19 - 26%).
  • A direct influence of the O2 concentration on induction of peroxide production occurred after reoxygenation with pure oxygen.
  • Increasing FCCP above 0.5 ยตM reduced the oxygen flux (2.7 โ€“ 6.6%) and the peroxide production (3.1 to 5 fold; Fig. 9A 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 ยตM 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, however, not as strong as observed with permeabilized fibres.
  • Generalized analysis of the experiments is difficult because of the effects of O2 dependence.


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