mitochondria respiration: low state 3, high ADP to Oxygen ratio
Brad Huffington
Wednesday, 08 April 2009 00:05 UTC
Hi,
I’m trying to measure oxygen consumption in isolated mouse liver mitochondria. After much trouble, I now consistently isolate “functional” mitochondria judging by an obvious response to ADP and acceptable state 3/4 ratios.
My problems: State 3 oxygen consumption is ~ 2-4x lower than it should be. And more strangely, I frequently have impossibly high ADP:O ratios (for example, 3.5 with glutamate/malate as a substrate, 2.5 with succinate. And I’m fairly sure my calculations are correct). When I omit bovine serum albumin from the respiration buffer the ADP:O decreases to something more normal, but it also makes my state 3/4 ratio much worse.
I’m currently using the protocol from Nature Protocols, with mannitol instead of sucrose in the isolation buffer and BSA in the respiration buffer (http://www.natureprotocols.com/2007/02/22/organelle_isolation_functional.php).
Any thoughts or suggestions would be appreciated.
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Replies
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Hi, Are you performing a percoll gradient or using crude mitochondria?
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Crude mitochondria. 600g, then 7000 g to pellet mitochondria and wash.
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Hi,
What is your RCRs values.
I can guess you have high ADP/O ratios because of your high state 4. Check if you can improve it with Oligomycin. -
Anonymous
Hi Sergei,
Yesterday, with glutamate/malate as substrate, I had an RCR of 6 and an ADP:O ratio of 4. With succinate, I had an RCR of 16.7 and an ADP:O of 2.8. So my state 4 is pretty low. Oligomycin might decrease it even further, but then I would not be able to calculate ADP:O. I’ve found that adding 0.1% fatty-acid free BSA to the respiration buffer virtually eliminates state 4 respiration (while increasing state 3), but it increases the ADP:O ratio even more.
Also, in my experiments the concentration of mitochondrial protein has an effect. More mitochondria seems to decrease ADP:O to a “normal” value, but it also decreases RCR (and means that I don’t have enough mitochondria for all my experiments). Does anyone know if this is normal?
Brad
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Hi, I am also doing isolation mitochondria from cancer cells according to this protocol. The RCR I got only 1.7. I used 300mM mannitol instead of sucrose in IB , as for EB, something is missing in the protocol, but I use 100 mM of mannitol and 0.1% BSA. Anyone has any clue what’s wrong or just because of the cancer cells.
Thanks a lot.
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mqin qin,
For the EB, it looks like the authors made a mistake in the preparation instructions (the paper could have used some editing: they even spelled “fibroblasts” wrong in the title). I found the correct recipe from one of the earlier papers by the authors of the review.
For your cells, don’t cancer cells have a decreased rate of oxidative phosphorylation? I don’t know if it the RCR would be affected, but it’s possible. But you should still have controls to reassure yourself your protocol is working and your mitochondria are intact.
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Dear All
please accept the apologies of us authors of this protocol for the small mistakes in the editing caused by poor typesetting of our paper. Indeed there are no filroblasts, but fibroblasts in the title. As for the recipe of the EB
(mix 12.5 ml of 1 M KCl, 1 ml of 1 M Tris/MOPS, 10 ml of 100 mul 0.1 M EGTA/Tris and 100 mul of Pi. Adjust pH to 7.4. Bring the volume to 100 ml with distilled water), undoubtedly one can’t add 10 mL of 100 muL EGTA-Tris (this makes no sense) but should add only 100 muL of EGTA-Tris. Again, apologies for the poor typesetting also here.
Going back to the problems of Brad and Mqin on I don’t believe that an ADP:O ratio of 3.5 and 2.5 for G/M and succinate are unrealistic for mouse liver mitochondria; similarly, an RCR below 2 is often reported in the case of mitochondria isolated from cells. The beautiful RCR by Brad actually reflect perfectly coupled mitochondria and are well in accordance with the stoichiometry of the respiratory chain. As for the low RCR in cancer cells, the most common problem here is that mitochondria are of poor quality due to dilution during isolation. Try to start from a higher amount of cells and your RCR will increase. Similarly, try to add 10 muM cytochrome c in the EB buffer to compensate for broken mitochondria in the sample. -
Dear Prof. Scorrano,
Thank you for contributing to our forum and for highlighting the mistake in the reagents – we will try to get this corrected in the original article as soon as possible. Unfortunately we will not be able to change the typo in the title – many apologies about this. Hopefully everyone will be able to work out that it means fibroblasts.
Best wishes,
Bronwen -
Dear guys! sorry for the typo(s) in the protocol and thanks for letting us know.
Just a few comments:
cancer mitochondria may be very delicate and due to the presence of lipid (in some cancer cells there is an increase in lipid synthesis) they may undergo damages of the membranes.
BSA may help in preventing the damages of lipids but may then cause some artifacts as you noticed.
The defects in the OXPHOS in cancer cells may be due to damages at the respiratory chain complexes or due to uncoupling or to problems at the TCA cycle so you can expect very weird results accordingly
Do you guys calculate state 4 after all the ADP has been consumed or before its addition?
Just to make sure that the EB will be OK, remember that the final concentration of EGTA should be 10 micromolar!
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