Talk:MiPNet17.08 IOC68 Schroecken AT: Difference between revisions
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== IOC68 Workshop Experiments == Β | == IOC68 Workshop Experiments == Β | ||
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* Oxygraph AB (in both chambers the same SUIT protocol was applied) | * Oxygraph AB (in both chambers the same SUIT protocol was applied) | ||
* DatLab File 2012-04-14_AB-02 | * 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 | * 2.3 mg tissue per chamber = 1.15 mg / ml | ||
* Flux derivation = 20 | * 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 + H<sub>2</sub>O<sub>2</sub> + D2 + D3 + S + FCCP + Rot + H<sub>2</sub>O<sub>2</sub> + H<sub>2</sub>O<sub>2</sub> + Mna + Ama | * '''SUIT-Protocol''': PMG +AmR+HRP +H<sub>2</sub>O<sub>2</sub> +D2 +D3 +S +U(FCCP) +Rot +H<sub>2</sub>O<sub>2</sub> +H<sub>2</sub>O<sub>2</sub> +Mna +Ama | ||
* | * The homogenate was added, the oxygen concentration was increased up to 320 nmol/ml and then the O2k-chamber was closed. | ||
=== Notes === | === Notes === | ||
Β Β Β | Β Β Β | ||
* following states were obtained: Β | * The following respiratory states were obtained: Β | ||
:: CI-linked | :: 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 | :: ADP-stimulated state ''P'' after titration of 3 mM ADP. | ||
:: CI | :: CI<small>&</small>II-linked respiration after titration of 10 mM succinate. | ||
:: | :: Noncoupled state ''E'' at 0.5 Β΅M FCCP. | ||
:: S(Rot) with 0.5 Β΅M rotenone. | :: S(Rot) (CII-linked) state ''E'' with 0.5 Β΅M 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 H<sub>2</sub>O<sub>2</sub> flux was reduced by about 2.6 to 9.2%. | ||
* | * Reoxygenation with pure oxygen increased the O<sub>2</sub> flux (14 - 18%) as well as the H<sub>2</sub>O<sub>2</sub> flux (19 - 26%). | ||
* | * A direct influence of the O<sub>2</sub> concentration on induction of peroxide production occurred after reoxygenation with pure oxygen. Β | ||
* FCCP reduced the oxygen flux (2.7 β 6.6 %) and the peroxide production (3.1 to 5 fold; | * Increasing FCCP above 0.5 Β΅M reduced the oxygen flux (2.7 β 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 C<sub>I</sub> was added, which reduced the oxygen flux (2 fold decrease) as well as the H<sub>2</sub>O<sub>2</sub> flux (25 - 29%) in both chambers. | ||
* | * In both chambers, the highest H<sub>2</sub>O<sub>2</sub> / O<sub>2</sub> flux ratio (''L'' with PMG) was 0.0034 (0.34%) and the addition of ADP diminished the H<sub>2</sub>O<sub>2</sub> / O<sub>2</sub> flux ratio to 0.0010 (0.10%). | ||
* | * Titration of succinate further diminished the ratio to 0.0007 (0.07%) and 0.5 Β΅M FCCP resulted in a H<sub>2</sub>O<sub>2</sub> / O<sub>2</sub> 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 H<sub>2</sub>O<sub>2</sub> 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. | ||
* analysis of the experiments is difficult because of O<sub>2</sub> dependence | * Generalized analysis of the experiments is difficult because of the effects of O<sub>2</sub> dependence. | ||
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Latest revision as of 10:27, 22 July 2019
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 Β΅M FCCP.
- S(Rot) (CII-linked) state E with 0.5 Β΅M 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 Β΅M reduced the oxygen flux (2.7 β 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 Β΅M 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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