Difference between revisions of "Talk:MiPNet17.08 IOC68 Schroecken AT"

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 µM FCCP.

S(Rot) (CII-linked) state E with 0.5 µM 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 µM reduced the oxygen flux (2.7 – 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_I 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, 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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@@ Line 9: / Line 9: @@
 * 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 till 320 nmol/ml and then the chamber was closed
+* The homogenate was added, the oxygen concentration was increased up to 320 nmol/ml and then the O2k-chamber was closed.
 === Notes ===
@@ Line 16: / Line 16: @@
 * 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 D after titration of 3 mM ADP
+:: ADP-stimulated state ''P'' after titration of 3 mM ADP.
-:: CI<small>&</small>II-linked respiration after titration of 10 mM succinate
+:: CI<small>&</small>II-linked respiration after titration of 10 mM succinate.
-:: Noncoupled state with 0.5 µM FCCP
+:: Noncoupled state ''E'' at 0.5 µM FCCP.
-:: S(Rot) (CII-linked) with 0.5 µM rotenone.
+:: S(Rot) (CII-linked) state ''E'' with 0.5 µM rotenone.
-* Titration of 3 mM ADP stimulated the oxygen flux about 69 to 73 % and inhibited the peroxide production 1.0 to 4.7%.
+* 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 about 27 % whereas H<sub>2</sub>O<sub>2</sub> flux was reduced about 2.6 to 9.2%.
+* 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 peroxide production as an induction of peroxide production occurred after reoxygenation with pure oxygen.
+* A direct influence of the O<sub>2</sub> concentration on 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 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.
+* Increasing FCCP above 0.5 µM reduced the oxygen flux (2.7 – 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%)
+* 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 µM 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 not that strong as observed with fibres.
+* 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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