Assay of microsomal oxysterol 7alpha-hydroxylase activity in the hamster liver by a sensitive method: in vitro modulation by oxysterols.
A method of assaying hepatic cytochrome P-450, oxysterol 7alpha-hydroxylase (CYP7B), was developed by combining the use of 25-[26,27-(3)H]hydroxycholesterol as a substrate and hydroxypropyl-beta-cyclodextrin as a substrate vehicle. When these assay conditions were tested, an undesirable transformation was observed of the reaction product, 7alpha,25-dihydroxycholesterol, into 3-oxo-7alpha,25-dihydroxy-4-cholesten by the activity of 3beta-hydroxy-Delta(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 7alpha-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 7alpha-hydroxycholesterol (-21%), 7beta-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.
Souidi
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M
Laboratoire de Physiologie de la Nutrition, Unité Associée Université Paris Sud/INRA, Bâtiment 447, Université Paris Sud, 91405 Orsay, Cedex, France.
Parquet
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M
Dubrac
S
S
Audas
O
O
Bécue
T
T
Lutton
C
C
D000818:Animals; D002784:Cholesterol / Q000494:pharmacology; D002790:Cholesterol 7-alpha-Hydroxylase / Q000032:analysis; D006224:Cricetinae; D016572:Cyclosporine / Q000494:pharmacology; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis / Q000378:metabolism*; D005954:Glucosephosphate Dehydrogenase; D006888:Hydroxycholesterols / Q000494:pharmacology; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP; D013250:Steroid Hydroxylases / Q000032:analysis / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014316:Tritium
D065607:Cytochrome P-450 Enzyme Inhibitors; D006888:Hydroxycholesterols; D014316:Tritium; D009249:NADP; D016572:Cyclosporine; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D005954:Glucosephosphate Dehydrogenase; D013250:Steroid Hydroxylases; C115867:oxysterol 7-alpha-hydroxylase; D002790:Cholesterol 7-alpha-Hydroxylase