Assessment of mitochondrial respiratory chain function.

Rat hearts were aerobically perfused with/without Intralipid (1%). Complex I, complex II, and complex IV function was measured polarographically by monitoring oxygen consumption in presence of specific substrates and ADP in saponin-skinned cardiac fibers using high-resolution respirometry (HRR). Complex III enzymatic activity was assayed spectrophotometrically (SP). The measured oxygen consumption (normalized to citrate synthase activity) is expressed as nmol O2*s−1/CS. Complex III activity is expressed as mOD*min−1/µg mitochondrial protein. Data are presented as mean (SEM). N = 6–10 in all groups. Abbreviations: AER, aerobically perfused hearts without treatment; AER/IL, aerobically perfused hearts exposed to Intralipid (1%); TMPD, tetramethyl-p-phenylene diamine, an artificial electron carrier which is reduced by ascorbate producing electrons that are transferred to cytochrome c; OD, optical density.