Improved assay of hepatic microsomal cholesterol 7 alpha-hydroxylase activity by the use of hydroxypropyl-beta-cyclodextrin and an NADPH-regenerating system

Clinica Chimica Acta; International Journal of Clinical Chemistry
M SouidiC Lutton

Abstract

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 formatio...Continue Reading

References

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Oct 13, 2006·Current Opinion in Gastroenterology·Hideyuki HyogoDavid E Cohen

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Citations

Feb 28, 2006·Archives of Toxicology·E TissandieM Souidi
May 4, 2002·The Journal of Nutritional Biochemistry·Murielle M.J. Combettes-SouverainClaude Y. Lutton
Oct 23, 2009·The Journal of Biological Chemistry·Jenna K CapykWilliam W Mohn
May 13, 2004·The British Journal of Nutrition·Ofélia P BentoMichel Riottot
Nov 23, 2006·Biochimica Et Biophysica Acta·E TissandiéM Souidi
Mar 31, 2004·Canadian Journal of Physiology and Pharmacology·P ScanffP Gourmelon
Nov 14, 2003·Canadian Journal of Physiology and Pharmacology·Maâmar SouidiClaude Lutton
Jan 5, 2002·The Journal of Nutrition·Zesheng ZhangZhen-Yu Chen

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