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| Outer mitochondrial membrane damage can be concluded from stimulation of respiration by exogenously added cytochrome ''c''.
| | #REDIRECT [[Cytochrome c]] |
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| == Determination of cytochrome ''c'' loss ==
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| Adding cytochrome ''c'' (10 µM; see [[Gnaiger_2002_BiochemSocTrans]]) to stimulate respiratory activity provides an estimate of cytochrome ''c'' loss. In general, maximum respiratory activity is obtained under conditions of saturating substrate concentrations and system dependent in the coupled (ADP stimulated) [[OXPHOS|State ''P'']] or non-coupled (uncoupler stimulated) [[ETS|State ''E'']].
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| ===Cytochrome c test===
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| When using cytochrome ''c'' as a quality control for permeabilised muscles from various species, which cytochrome ''c'' should we use (a wide range of types of cytochrome ''c'' is available from Sigma-Aldrich) and at which concentration?
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| We apply routinely cytochrome ''c'' from Sigma C 7752 (see MiPNet03.02-Selected Media and Chemicals). It would be interesting to compare the consequence of application of different sources of cytochrome ''c''.
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| A detailed discussion on the dependence of respiration in isolated mitochondria and permeabilized cardiac fibers is found in refs. 1 and 2, for the first time showing that cytochrome ''c'' kinetics is different when studying a segment of the [[ETS]] ([[CII]]-linked: succinate+rotenone) versus the isolated step of cytochrome ''c'' oxidase ([[CIV]]; ascorbate+TMPD+antimycin A). For CII-linked respiration, an exernal cytochrome ''c'' concentration of 10 µM yields kinetic saturation (monophasic hyperbolic), but kinetics is biphasic for CIV and 10 µM is not saturating (ref. 1 and 2).
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| Importantly, cytochrome ''c'' increases the chemical background oxygen flux in the presence of ascorbate and TMPD, dependent on oxygen concentration and cytochrome ''c'' concentration, and appropriate chemical background corrections are required (ref. 3). Without ascorbate and TMPD, added cytochrome ''c'' is stable.
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| Evaluation of the cytochrome ''c'' effect, when respiration is slightly unstable:
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| Mark respiration just before cytochrome ''c'' addition and after. Take these to values to calculate the increase of respiration due to cytochrome ''c'' addition.
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| ===At which step of the protocol should cytochrome ''c'' be added? ===
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| If a stimulatory effect of cytochrome ''c'' is observed, respiratory capacities measured before cytochrome ''c'' addition might be cytochrome ''c'' limited and therefore underestimated. This provides an argument to add cytochrome ''c'' at an early state of the protocol.
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| It is important '''not''' to add cytochrome ''c'' in a [[LEAK state]]: There is always an unexplained activation of respiration, unrelated to the injury of the outer mt-membrane. Add cytochrome ''c'' only after activation by ADP.
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| == Cytochrome ''c'' release ==
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| === Cytochrome ''c'' release induced by sample preparation ===
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| A preparation induced damage of the outer mitochondrial membrane and as a result subsequent loss of cytochrome ''c'' can be detected by a stimulation of respiration after the addition of cytochrome ''c''.
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| The preparation induced damage can also affect the respiratory complexes. Therefore, experimental runs showing a preparation induced cytochrome ''c'' release should be excluded from the final data set.
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| In perfectly prepared muscle fibers cytochrome ''c'' should have no stimulatory effect on maximum respiratory activity, in liver biopsies a small effect is observed, even in carefully prepared samples.
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| === Cytochrome ''c'' release induced by treatment ===
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| Treatment-triggered cytochrome ''c'' release, e.g. cell death induction, has to be distinguished from preparation induced damage.
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| If cytochrome ''c'' is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result.
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| ==References==
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| #[[Gnaiger_2002_BiochemSocTrans|Gnaiger E, Kuznetsov AV (2002) Mitochondrial respiration at low levels of oxygen and cytochrome c. Biochem. Soc. Trans. 30: 242-248.]] | |
| #[[Kuznetsov_2004_AJP|Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641.]]
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| *See also: [[Renner_2003_BBA|Renner K, Amberger A, Konwalinka G, Kofler R, Gnaiger E (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim. Biophys. Acta 1642: 115-123.]]
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| *[[Cytochrome c]]
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| *[http://www.oroboros.at/index.php?cytochromeccontrol Cytochrome c Control]
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| {{#set: Scientific service =Quality Control}}
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| __SHOWFACTBOX__
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| {{Scientific service}}
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