= Isolated mitochondria respiration experiment
<Respiratory control> can be investigated outside an intact <biological cell> by supplying isolated <mitochondria> with <substrates> and <ADP>. This makes the boundary between <oxygen> consumption and <ATP> formation experimentally accessible. The historical <mitochondrial oxygen electrode assay> describes the 1955 experiment; the following authored protocol adds modern preparation and inhibitor controls.
In Functional Assessment of Isolated Mitochondria In Vitro (2009), <Ian R. Lanza> and <K. Sreekumaran Nair> describe human skeletal-muscle biopsy mincing, cold <protease> treatment, homogenization and <differential centrifugation>. A low-speed spin removes coarse material; subsequent spins collect and wash <mitochondria>. A stirred, closed $2\,\mathrm{mL}$ Oxygraph-2k chamber at $37\,{}^\circ\mathrm{C}$ uses a calibrated polarographic <oxygen> <electrode>. Its signal gives dissolved <oxygen> <concentration>; the negative time <derivative> gives consumption. Instrument corrections include electrode consumption and <oxygen> leakage.<Methods in Enzymology, volume 457: Mitochondrial Function, Part B/Functional Assessment of Isolated Mitochondria In Vitro>[Methods in Enzymology, volume 457: Mitochondrial Function, Part B; Tissue homogenization; Separation of mitochondria by differential centrifugation; Mitochondrial respiration, Overview; An oxygraph protocol for mitochondrial assessment, steps 1–2]{ref}
Their additions include <glutamate> plus <malate>, an <ADP> pulse, then saturating <ADP>. Added <cytochrome c> tests outer-membrane damage: stimulation suggests loss of endogenous <cytochrome c>. Later <oligomycin> inhibits <ATP synthase>; <FCCP> produces <respiratory uncoupling>; <antimycin A> inhibits <respiratory complex III> to estimate residual background. Figure 6 includes intervening <succinate dehydrogenase> and <respiratory complex I> interventions; the modeled traces below simplify that sequence. Isolation can damage or select <mitochondria>, and saturating assay conditions differ from those in living <biological tissue>.<Methods in Enzymology, volume 457: Mitochondrial Function, Part B/Functional Assessment of Isolated Mitochondria In Vitro>[Methods in Enzymology, volume 457: Mitochondrial Function, Part B; Figure 3; An oxygraph protocol for mitochondrial assessment, steps 3–11 and Figure 6; Summary, nonphysiological assay conditions]{ref}
The control logic matters more than a single steep slope. If <ADP> accelerates consumption and a finite pulse is followed by slowing, demand is affecting respiratory flux. If <oligomycin> slows consumption, but subsequent <FCCP> accelerates it, the <electron transport chain> can still turn over while <ATP synthase> remains inhibited. That contrast separates restricted coupled turnover from a completely inactive chain. Failure of <FCCP> to restore a rate is less specific: <substrate> limitation, damage, low <oxygen> or excessive uncoupler can also restrict respiration.
Neither the <cytochrome c> test alone nor a large <respiratory control ratio> establishes every aspect of mitochondrial integrity. Likewise, <oxygen> consumption is not a direct <ATP> assay: <respiratory uncoupling> provides a concrete counterexample. The preparation, additions and normalization denominator determine what comparison the experiment can support.
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