Cholesterol and 27-hydroxycholesterol 7 alpha-hydroxylation: evidence for two different enzymes.
The use of 2-hydroxypropyl-beta-cyclodextrin as a vehicle for solubilizing cholesterol and 27-hydroxycholesterol has led to a study of their rates of 7 alpha-hydroxylation in microsomal preparations from hamster liver and HepG2 cells. Addition of the vehicle alone to the cholesterol 7 alpha-hydroxylase assay always caused a several-fold increase in activity. Preloading the vehicle with cholesterol further augmented the rate of 7 alpha-hydroxycholesterol formation. Preloading the vehicle with 27-hydroxycholesterol or 27-hydroxycholestanol (molar ratio 1/1.2) minimally decreased cholesterol 7 alpha-hydroxylase activity (-12%), compared with preloading with cholestanol (-50%), a known competitive inhibitor of the enzyme. Microsomes from hamster liver yielded rates of 7 alpha,27-dihydroxcholesterol formation of 1.5 to 3.0 nmol/min per mg protein, compared with 0.3 nmol/min per mg protein for 7 alpha-hydroxycholesterol. Although cholesterol and cholestanol had minimal effects on the rate of 7 alpha-hydroxylation of 27-hydroxycholesterol, addition of an approximately equimolar amount of 27-hydroxycholestanol inhibited the rate of formation by 65%. Attempts to separate and identify the two C-27 sterol 7 alpha-hydroxylases chromatographically led to the finding that Emulgen 913 selectively inactivates 7 alpha-hydroxylation of 27-hydroxycholesterol. These results indicate that the metabolic pathway for bile acid synthesis from 27-hydroxycholesterol is not governed by cholesterol 7 alpha-hydroxylase.
Division of Hepatic Diseases, New York University Medical Center, NY 10016.
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Martin
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Budai
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Javitt
D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D003505:Cyclodextrins / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D006801:Humans; D006888:Hydroxycholesterols / Q000378:metabolism*; D006900:Hydroxylation; D066298:In Vitro Techniques; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism; D013250:Steroid Hydroxylases / Q000378:metabolism*; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D006888:Hydroxycholesterols; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; C479658:hydroxypropyl-alpha-cyclodextrin; D000073738:2-Hydroxypropyl-beta-cyclodextrin; C076996:27-hydroxycholesterol; D003577:Cytochrome P-450 Enzyme System; D002784:Cholesterol; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; C029009:steroid hormone 7-alpha-hydroxylase