Assay of microsomal oxysterol 7α-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols

• Published in: Biochimica et biophysica acta. Molecular and cell biology of lipids Vol. 1487; no. 1; pp. 74 - 81
• Main Authors: Souidi, Maâmar, Parquet, Michel, Dubrac, Sandrine, Audas, Olivier, Bécue, Thierry, Lutton, Claude
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 24.08.2000
• Subjects: 7α-Hydroxycholesterol; Cholesterol 7α-hydroxylase; Epicoprostanol; Hydroxypropyl-β-cyclodextrin; Oxysterol 7α-hydroxylase; Sterol 27-hydroxylase; Cholesterol 7α-hydroxylase; CYP27, sterol 27-hydroxylase; CYP8B, sterol 12α-hydroxylase; Oxysterol 7α-hydroxylase; CYP7A, cholesterol 7α-hydroxylase; Sterol 27-hydroxylase; Epicoprostanol; Hydroxypropyl-β-cyclodextrin; CYP7B, oxysterol 7α-hydroxylase; 7α-Hydroxycholesterol; HPBCD, hydroxypropyl-β-cyclodextrin
• ISSN: 1388-1981; 1879-2618
• DOI: 10.1016/S1388-1981(00)00086-X

A method of assaying hepatic cytochrome P-450, oxysterol 7α-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27- 3H]hydroxycholesterol as a substrate and hydroxypropyl-β-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7α,25-dihydroxycholesterol, into 3-oxo-7α,25-dihydroxy-4-cholesten by the activity of 3β-hydroxy-Δ 5-C 27 steroid oxydoreductase, a microsomal NAD + and NADP + dependent enzyme of bile acid metabolism. A great improvement was reached by using a continuous NADPH generating system which constantly re-transforms NADP + into NADPH, thus inhibiting this activity. This improved CYP7B assay, comparable to our previously described assay for cholesterol 7α-hydroxylase (CYP7A), allowed a 3-fold increase of the apparent enzyme activity. The possibility to simultaneously measure CYP7A and CYP7B activities on the same microsomal preparation was investigated. A marked decrease (−33%) in the CYP7B activity was noticed, while that of CYP7A remained unchanged. The CYP7B activity was observed to be inhibited by cholesterol (−30%) and also by the oxysterols 7α-hydroxycholesterol (−21%), 7β-hydroxycholesterol (−25%) and epicoprostanol (−20%), and by cyclosporin A (−26%). It can be concluded that this sensible and easy to perform CYP7B assay allows to observe, at least in vitro, a modulation of the enzyme activity by oxysterols.