Table 1.

Overview of the assays currently in use in the Nijmegen Center for mitochondrial disorders

Enzyme	Substrate	Assay conditions	Read-out
Complex I	0.2 mmol/L NADH	25 mM phosphate buffer, pH 7.6	1 µmol/L rotenone-sensitive DCIP reduction at 600 nm
	70 µmol /L coenzyme Q1	0.35% BSA
		60 µmol/L DCIP
Complex II	10 mmol/L succinate	80 mM phosphate buffer, pH 7.8	5 mmol/L malonate-sensitive DCIP reduction at 600 nm
	80 µmol/L decylubiquinone	0.2 % BSA
		0.2 mmol/L ATP
		80 µmol/L DCIP
		2.0 mmol/L EDTA
		0.3 mmol/L sodium azide
Complex III	300 µmol/L decylubiquinol	50 mM phosphate buffer, pH 7.8	Cytochrome c reduction at 550 nm
	50 µmol/L cytochrome c	1.0 mmol/L EDTA
		3.0 mmol/L sodium azide
		0.04% Tween20
Complex IV	70 µmol/L reduced cytochrome c	30 mM phosphate buffer, pH 7.4	Cytochrome c oxidation at 550 nm
Complex V	3 mmol/L ATP	25 mM phosphate buffer, pH 8.0	10 µmol/L oligomycin-sensitive NADH oxidation at 340 nm
		0.2 mmol/L EGTA
		5 mg/L Ap5A
		0.3% BSA
		250 mmol/L sucrose
		7.5 mmol/L MgCl2
		50 mmol/L KCl
		0.1 mmol/L phosphoenolpyruvate
		2.5 U/ml lactate dehydrogenase
		1.5 U/ml pyruvate kinase

The assays are performed at 37°C. The samples that are tested with these assays include isolated mitochondria from muscle and fibroblasts. In addition, more crude preparations from muscle, such as 10% homogenates in SETH-buffer (Janssen et al 2006), and the 600-g supernatants of these homogenates, are measured in this way. Extracts from heart, liver, brain, and chorionic villi are examined in a similar manner. These assays can be performed on any spectrophotometric device that has sufficient sensitivity.