Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system.
Cholesterol 7 alpha-hydroxylase, the key enzyme in bile acid synthesis, has been implicated in atherosclerosis and gallstone disease. The aim of this study was to check if the use of hydroxypropyl-beta-cyclodextrin (HPBCD), a vehicle for solubilizing cholesterol, augmented the rate of 7 alpha-hydroxycholesterol formation in hamster liver microsomes compared to classical assays in which labeled cholesterol was delivered in Tween 80. We observed that [14C]cholesterol carried by HPBCD enhanced the sensitivity of the assay tenfold. However, linearity of 7 alpha-hydroxycholesterol formation with time was short because of the rapid transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one and 7 alpha,12 alpha-dihydroxy-cholesten-3-one when NADPH alone was present in the incubation medium. In order to avoid the transformation of 7 alpha-hydroxycholesterol into 7 alpha-hydroxy-cholesten-3-one, which is essentially NAD(+)-dependent, but is also NADP(+)-dependent, NADPH (1 mmol/l) plus an NADPH-regenerating system must be present in the medium. In this improved assay, the optimal pH was 7.4 and the apparent Km for control and cholestyramine-fed hamsters had a similar value of 315 mumol/l; linearity in the formation of 7 alpha-hydroxycholesterol was also apparent after a relatively short time period (10 min), but with a markedly greater slope of the curve. With a short incubation time (6 min), microsomes from livers of hamsters (five and nine weeks old) that were fed with a commercial ground diet yielded rates of 7 alpha-hydroxycholesterol formation of 115 +/- 10 and 150 +/- 16 pmol/min.mg protein, respectively, whereas microsomes from hamsters fed with a lithogenic sucrose-rich diet (five weeks old) yielded rates of 7 alpha-hydroxycholesterol formation of 77 +/- 7 pmol/min.mg protein, which were significantly lower (-33%) than those of corresponding control hamsters. This improved cholesterol 7 alpha-hydroxylase assay is very sensitive, simple and rapid, and does not necessitate sophisticated equipment. It can be particularly useful for determining cholesterol 7 alpha-hydroxylase activity in liver biopsies from dyslipidemic or lithiasic patients.
Souidi
M
M
Laboratoire de Physiologie de la Nutrition, Université Paris-Sud, Orsay, France.
Parquet
M
M
Lutton
C
C
D000073738:2-Hydroxypropyl-beta-cyclodextrin; D000367:Age Factors; D000818:Animals; D002790:Cholesterol 7-alpha-Hydroxylase / Q000378:metabolism*; D006224:Cricetinae; D003505:Cyclodextrins / Q000494:pharmacology*; D004032:Diet; D004229:Dithiothreitol / Q000494:pharmacology; D008297:Male; D008647:Mesocricetus; D008862:Microsomes, Liver / Q000201:enzymology*; D009249:NADP / Q000378:metabolism*; D011136:Polysorbates / Q000494:pharmacology; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins
D003505:Cyclodextrins; D011136:Polysorbates; D047391:alpha-Cyclodextrins; D047392:beta-Cyclodextrins; C479658:hydroxypropyl-alpha-cyclodextrin; D000073738:2-Hydroxypropyl-beta-cyclodextrin; D009249:NADP; D002790:Cholesterol 7-alpha-Hydroxylase; D004229:Dithiothreitol