RCR is low in isolated mitochondria: Difference between revisions

Revision as of 18:22, 13 November 2011

Problem

At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly if added at a concentration typically used for State 3 measurements (200-300 µM), which may stimulate flux below the ADP-saturated state of OXPHOS capacity. The slope increases sharply and returns to State 4 (LEAK state), even before the plot of flux shows the true maximum, resulting in a low apparent RCR.

Tests and Solutions

Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the Oxygraph Control window; see MiPNet12.06 O2k-Start). As the data recording interval is reduced, the flux appears more noisy, but represents transitions more accurately and reduces the apparent time-delay. At high flux per volume, the increased noise level presents no problem. Flux then shows a period of constant OXPHOS capacity rather than a sharp peak.

Reduce the mitochondrial concentration, thus prolonging the duration of ADP-saturated respiration (OXPHOS capacity) over 120 seconds. If flux reaches a constant maximum value, then analysis is possible without application of signal deconvolution (standard plot of flux in DatLab).

Otherwise, export the data into DatLab 2, and apply a time correction (signal deconvolution), as described by Gnaiger 2001 RespPhysiol.

See also ‘Notes on Time resolution’.

Template:Troubleshooting

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 == Problem ==
-At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly. The slope increases sharply and returns to State 4 (LEAK state), even before the plot of flux shows the true maximum value of OXPHOS capacity (State 3), resulting in a low apparent RCR.
+At a high mitochondrial concentration (0.5 mg protein/ml), ADP is exhausted very rapidly if added at a concentration typically used for [[State 3]] measurements (200-300 µM), which may stimulate flux below the ADP-saturated state of [[OXPHOS capacity]]. The slope increases sharply and returns to State 4 (LEAK state), even before the plot of flux shows the true maximum, resulting in a low apparent RCR.
 == Tests and Solutions ==
-# Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the Oxygraph Control window; see [[MiPNet12.06 O2k-Start]]). As the data recording interval is reduced, the flux appears more noisy, but represents transitions more accurately and reduces the apparent time-delay. At high flux per volume, the increased noise level presents no problem. Flux then shows a period of constant state 3 respiration rather than a sharp peak, and routine analysis is possible in DatLab 4.
+# Reduce the data recording interval (standard is 2 seconds) to the minimum of 0.2 s (setting in the Oxygraph Control window; see [[MiPNet12.06 O2k-Start]]). As the data recording interval is reduced, the flux appears more noisy, but represents transitions more accurately and reduces the apparent time-delay. At high flux per volume, the increased noise level presents no problem. Flux then shows a period of constant OXPHOS capacity rather than a sharp peak.
 # Reduce the mitochondrial concentration, thus prolonging the duration of ADP-saturated respiration (OXPHOS capacity) over 120 seconds. If flux reaches a constant maximum value, then analysis is possible without application of signal deconvolution (standard plot of flux in DatLab).
 # Otherwise, export the data into [http://www.oroboros.at/index.php?o2k-datlab2 DatLab 2], and apply a time correction (signal deconvolution), as described by [[Gnaiger 2001 RespPhysiol]].
 # See also ‘Notes on [[Time resolution]]’.