Table 2.

Enzymatic analysis of the different mitochondrial fatty acid beta-oxidation enzymes and the experimental conditions used in our laboratory for each of these enzymes

Enzyme/transporter	Assay medium	Incubation period and temperature	Method of analysis	Reference
CPT1	150 mmol/L KCl, 25 mmol/L Tris, 2 mmol/L EDTA, 20 mmol/L potassium phosphate, 1 mg/mL bovine serum albumin, 4.5 mmol/L GSH, 5 mmol/L KCN, 50 μg/mL digitonin, 0.5 mmol/L carnitine, pH = 7.0, plus 25 μmol/L [U-13C]-palmitoyl-CoA	10 min, 37°C	Tandem-MS	(van Vlies et al. 2007)
CACT	150 mmol/L KCl, 25 mmol/L Tris-HCl, 2.0 mmol/L EDTA, 10 mmol/L potassium phosphate, 10 mg/mL bovine serum albumin, 40 μg/mL digitonin, pH = 7.4 plus 0.1 mmol/L acetylcarntinine and [1-14C]-acetylcarnitine	30 min, 25°C	Radiometry (14CO2 release)	(IJlst et al. 2001)
CPT2	100 mmol/L Tris-HCl, 120 mmol/L KCl, 5 mmol/L GSH, 8 mmol/L CoASH, 2 mmol/L palmitoyl-carnitine, 0.2% (w/v) Triton X-100, pH = 7.0	20 min, 37°C	HPLC-UV	To be published
VLCAD	125 mmol/L Tris-HCl, 0.4 mmol/L ferricenium hexa-fluorophosphate, 0.25 mmol/L palmitoyl-CoA, pH 8.0	5 min, 37°C	HPLC-UV	To be published
MCAD	200 mmol/L Tris-HCl, 0.22 mmol/L 3-phenylpropionyl-CoA, 0.4 mmol/L ferricenium hexafluorophosphate, pH = 8.0	10 min, 37°C	HPLC-UV	To be published
LCHAD	100 mmol/L potassium phosphate, 50 mmol/L MOPS, 0.1 mmol/L DTT, 0.1% (w/v) Triton X-100, 0.15 mmol/L NADH, 5 mmol/L N-ethylmaleimide (if added), pH = 6.16, plus 50 μmol/L 3-ketopalmitoyl-CoA	Continuous assay, 37°C	Spectrophotometry (340 nm)	(Wanders et al. 1990)
LCTH	100 mmol/L Tris-HCl, 10 mmol/L MgCl2, 50 μmol/L CoASH, pH = 8.05, plus 50 μmol/L 3-ketopalmitoyl-CoA	Continuous assay, 37°C	Spectrophotometry (303 nm)	(Wanders et al. 1990)
SCHAD	100 mmol/L potassium phosphate, 50 mmol/L MOPS, 0.1 mmol/L DTT, 0.1% (w/v) Triton X-100, 0.15 mmol/L NADH, 5 mmol/L N-ethylmaleimide (if added), pH = 6.16, plus 50 μmol/L acetoacetyl-CoA	Continuous assay, 37°C	Spectrophotometry (340 nm)	(Wanders et al. 1990)