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A new mechanism for formation of 7-ketocholesterol was recently described involving cytochrome P-450 (CYP)7A1-catalyzed conversion of 7-dehydrocholesterol into 7-ketocholesterol with cholesterol-7,8-epoxide as a side product. Some patients with cerebrotendinous xanthomatosis (CTX) and all patients with Smith-Lemli-Opitz syndrome (SLO) have markedly increased levels of 7-dehydrocholesterol in plasma and tissues. In addition, the former patients have markedly upregulated CYP7A1. We hypothesized that these patients may produce 7-ketocholesterol from 7-dehydrocholesterol with formation of cholesterol-7,8-epoxide as a side product. In accord with this hypothesis, two patients with CTX were found to have increased levels of 7-ketocholesterol and 7-dehydrocholesterol, as well as a significant level of cholesterol-7,8-epoxide. The latter steroid was not detectable in plasma from healthy volunteers. Downregulation of CYP7A1 activity by treatment with chenodeoxycholic acid reduced the levels of 7-ketocholesterol in parallel with decreased levels of 7-dehydrocholesterol and cholesterol-7,8-epoxide. Three patients with SLO were found to have markedly elevated levels of 7-ketocholesterol as well as high levels of cholesterol-7,8-epoxide. The results support the hypothesis that 7-dehydrocholesterol is a precursor to 7-ketocholesterol in SLO and some patients with CTX.
Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.
7-Ketocholesterol is a bioactive sterol, a potent competitive inhibitor of cytochrome P450 7A1, and toxic in liver cells. Multiple origins of this compound have been identified, with cholesterol being the presumed precursor. Although routes for formation of the 7-keto compound from cholesterol have been established, we found that 7-dehydrocholesterol (the immediate precursor of cholesterol) is oxidized by P450 7A1 to 7-ketocholesterol (k(cat)/K(m) = 3 × 10(4) m(-1) s(-1)). P450 7A1 converted lathosterol (Δ(5)-dihydro-7-dehydrocholesterol) to a mixture of the 7-keto and 7α,8α-epoxide products (~1:2 ratio), with the epoxide not rearranging to the ketone. The oxidation of 7-dehydrocholesterol occured with predominant formation of 7-ketocholesterol and with the 7α,8α-epoxide as only a minor product; the synthesized epoxide was stable in the presence of P450 7A1. The mechanism of 7-dehydrocholesterol oxidation to 7-ketocholesterol is proposed to involve a Fe(III)-O-C-C(+) intermediate and a 7,8-hydride shift or an alternative closing to yield the epoxide (Liebler, D. C., and Guengerich, F. P. (1983) Biochemistry 22, 5482-5489). Accordingly, reaction of P450 7A1 with 7-[(2)H(1)]dehydrocholesterol yielded complete migration of deuterium in the product 7-ketocholesterol. The finding that 7-dehydrocholesterol is a precursor of 7-ketocholesterol has relevance to an inborn error of metabolism known as Smith-Lemli-Opitz syndrome (SLOS) caused by defective cholesterol biosynthesis. Mutations within the gene encoding 7-dehydrocholesterol reductase, the last enzyme in the pathway, lead to the accumulation of 7-dehydrocholesterol in tissues and fluids of SLOS patients. Our findings suggest that 7-ketocholesterol levels may also be elevated in SLOS tissue and fluids as a result of P450 7A1 oxidation of 7-dehydrocholesterol.
Cytochrome P450 (P450) 7A1 is well known as the cholesterol 7α-hydroxylase, the first enzyme involved in bile acid synthesis from cholesterol. The human enzyme has been reported to have the highest catalytic activity of any mammalian P450. Analyses of individual steps of cholesterol 7α-hydroxylation reaction revealed several characteristics of this reaction: (i) two-step binding of cholesterol to ferric P450, with an apparent K(d) of 0.51 μM, (ii) a rapid reduction rate in the presence of cholesterol (∼10 s(-1) for the fast phase), (iii) rapid formation of a ferrous P450-cholesterol-O(2) complex (29 s(-1)), (iv) the lack of a non-competitive kinetic deuterium isotope effect, (v) the lack of a kinetic burst, and (vi) the lack of a deuterium isotope effect when the reaction was initiated with the ferrous P450-cholesterol complex. A minimum kinetic model was developed and is consistent with all of the observed phenomena and the rates of cholesterol 7α-hydroxylation and H(2)O and H(2)O(2) formation. The results indicate that the first electron transfer step, although rapid, becomes rate-limiting in the overall P450 7A1 reaction. This is a different phenomenon compared with other P450s that have much lower rates of catalysis, attributed to the much more efficient substrate oxidation steps in this reaction.
Chemical derivatization is useful for improving the ionization characteristics of poorly or nonionizable analytes in liquid chromatography-mass spectrometry (LC-MS). Dansyl chloride has been widely used as a derivatizing reagent for fluorescence detection and for facilitating the MS detection of phenols and amines, but not for general alcohols. A new dansylation method for improving the mass spectral sensitivity of unactivated alcohols was developed. The dansylated derivative was formed after incubation of the test compound cholesterol and excess dansyl chloride in CH(2)Cl(2) in the presence of 4-(dimethylamino)-pyridine (DMAP) plus N,N-diisopropylethylamine at 65 °C for 1 h, with an overall yield of 96%. The versatility of dansylation was investigated by utilizing representative lipid compounds (containing different numbers of hydroxy groups) for dansylation. All dansylated derivatives of the selected compounds were detected by LC-MS/MS in the electrospray ionization (ESI) positive ion mode. Validation of the method was established in terms of the sensitivity, stability, and repeatability of dansylation. The method was then applied to characterizing the P450 7A1 oxidation product (dansylated 7α-hydroxycholesterol) in human liver extracts using an LC-MS metabolomics/isotopic labeling approach (Tang, Z.; Guengerich, F. P. Anal. Chem. 2009, 81, 3071-3078). The dansylated derivative of the product was identified, with the signal increased by 10(3)-fold compared with a previous method (derivatization with succinic anhydride and ESI negative ion MS). Quantitation of testosterone in human liver extracts was also done as an example of the application of the dansylation method. Thus, dansylation is a potential method of modifying many alcohols for detection by fluorescence and LC-MS analysis.