Assay of HMG-CoA and cholesterol 7α-hydroxylase enzyme activity
Mouse liver microsomes were prepared by differential centrifugation [33]. Briefly, 0.1–0.2 g liver was homogenized in a Potter-Elvjhem homogenizer with five volumes of buffer (0.25 M sucrose, 0.1 M Tris, 0.1 mM disodium EDTA, 0.1 mM DTT, pH 7.4). Microsomes were isolated by differential centrifugation (10,000 g to 100,000 g). The pellets were washed and resuspended in storage buffer (0.1 M dipotassium phosphate, 0.05 M KCl, 1 mM DTT, 5 mM disodium EDTA, 20% glycerol, pH 7.4) at 25–30 mg protein/ml. Protein concentrations were determined according to the method of Lowry et al. [34]. The activity of cholesterol 7α-hydroxylase (EC 1.14.12.17) was measured by an isotope incorporation method according to Shefer et al. [33] with some modifications. The reaction mixture (final volume 0.5 ml) consisted of potassium phosphate buffer (100 mM K2HPO4, 0.1 mM EDTA, 5 mM DTT, 30 mM nicotinamide, pH 7.4), [14C]-cholesterol (5 × 105 dpm) solubilized in 50 μl of 25% (wt/vol) β-cyclodextrin (final concentration 0.8%), and 50–200 μg of microsomal protein. The reaction was initiated by the addition of NADPH or an NADPH-generating system (3.4 mM NADP+, 30 mM glucose-6-phosphate, 0.3 U of glucose-6-phosphate dehydrogenase) and continued for 15 min at 37°C. The reaction was stopped with 0.5 ml of 1 N KOH, 5 μg of butylated hydroxytoluene, and 10 μl of ethanolic potassium hydroxide. [3H] 7α-hydroxycholesterol (1 × 104 dpm/5 μg) was added as an internal recovery standard. After saponification at 37°C for one hour, sterols were extracted twice with 3 ml of n-hexane and the extracts were evaporated to dryness under nitrogen. The residue was dissolved in 0.3 ml of n-hexane:2-propanol (97.3 vol/vol) and applied to a silica column (500 mg; sep-pak, by 4 ml of n-hexane:2-propanol (97.3 vol/vol)). 7α-hydroxycholesterol was eluted with 3 ml of n-hexane:2-propanol (80:20 vol:vol) and further isolated by TLC on silica gel plates (Silica gel 60, EM Science, Gibbestown, NJ, USA) with diethyl ether, and quantified by liquid scintillation counting using Ecolume (ICN Radiochemicals Irvine, CA, USA). Assays were carried out in duplicate with correction for zero-time controls. HMG-CoA reductase (EC 1.1.1.3.4) activity was determined according to the method of Xu et al. [35] with modifications as described [36].