Steatotic livers are susceptible to normothermic ischemia-reperfusion injury from mitochondrial Complex-I dysfunction

Table 3 Mitochondrial respiration assay protocol

Reagents added	Final concentration in oxygraph chamber (mmol/L)	Action of reagent	Measurement output
Step 1
Glutamate	10	CI substrates	CI leak respiration
Malate	5		(CILeak)
Pyruvate	10
Step 2
ADP	1.25	Substrate for ATP generation	CI oxidative phosphorylation
Step 3
Succinate	10	CII substrate	CI+CII oxidative phosphorylation1
Step 4
Rotenone	0.001	CI inhibitor	Isolate flux to CII [CII(rot)]
Step 5
Oligomycin	0.0025	ATP-Synthase inhibitor	CI+CII leak respiration (CI,IILeak)
Step 6
FCCP	0.0015	Mitochondrial uncoupler	ETS capacity
Step 7
Antimycin A	0.0050	CIII inhibitor	Residual oxidase consumption

¹The individual contribution of CII to oxidative phosphorylation (CII-OXPHOS) can also be derived (CI+II-OXPHOS minus CI-OXPHOS). ADP: Adenosine diphosphate; ATP: Adenosine triphosphate; CI: Complex I; CI-OXPHOS: Complex I oxidative phosphorylation; CI+II-OXPHOS: Complex I + Complex II oxidative phosphorylation; CI,II_Leak: Complex I + Complex II leak respiration; CI_Leak: Complex I leak respiration; CII: Complex II; CIII: Complex III; ETS: Electron transfer system; FCCP: Carbonylcyanide p-trifluoromethoxy-phenylhydrazone; Rot: Rotenone.