Novel oxysterols observed in tissues and fluids of AY9944-treated rats: a model for Smith-Lemli-Opitz syndrome
2011; Elsevier BV; Volume: 52; Issue: 10 Linguagem: Inglês
10.1194/jlr.m018366
ISSN1539-7262
AutoresLibin Xu, Wei Liu, Lowell G. Sheflin, Steven J. Fliesler, Ned A. Porter,
Tópico(s)Lipid metabolism and biosynthesis
ResumoTreatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ7-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d7-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.—. Treatment of Sprague-Dawley rats with AY9944, an inhibitor of 3β-hydroxysterol-Δ7-reductase (Dhcr7), leads to elevated levels of 7-dehydrocholesterol (7-DHC) and reduced levels of cholesterol in all biological tissues, mimicking the key biochemical hallmark of Smith-Lemli-Opitz syndrome (SLOS). Fourteen 7-DHC-derived oxysterols previously have been identified as products of free radical oxidation in vitro; one of these oxysterols, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), was recently identified in Dhcr7-deficient cells and in brain tissues of Dhcr7-null mouse. We report here the isolation and characterization of three novel 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC and 24-hydroxy-7-DHC) in addition to DHCEO and 7-ketocholesterol (7-kChol) from the brain tissues of AY9944-treated rats. The identities of these five oxysterols were elucidated by HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR. Quantification of 4α- and 4β-hydroxy-7-DHC, DHCEO, and 7-kChol in rat brain, liver, and serum were carried out by HPLC-MS using d7-DHCEO as an internal standard. With the exception of 7-kChol, these oxysterols were present only in tissues of AY9944-treated, but not control rats, and 7-kChol levels were markedly (>10-fold) higher in treated versus control rats. These findings are discussed in the context of the potential involvement of 7-DHC-derived oxysterols in the pathogenesis of SLOS.—. 7-Dehydrocholesterol (7-DHC) accumulates in tissues and fluids of patients with Smith-Lemli-Opitz syndrome (SLOS), a recessive disease caused by mutations in the gene encoding 3β-hydroxysterol-Δ7-reductase (DHCR7; EC 1.3.1.21), the enzyme that catalyzes the conversion of 7-DHC to cholesterol (1Irons M. Elias E.R. Salen G. Tint G.S. Batta A.K. Defective cholesterol biosynthesis in Smith-Lemli-Opitz syndrome.Lancet. 1993; 341: 1414Abstract PubMed Scopus (324) Google Scholar, 2Tint G.S. Irons M. Elias E.R. Batta A.K. Frieden R. Chen T.S. Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome.N. Engl. J. Med. 1994; 330: 107-113Crossref PubMed Scopus (682) Google Scholar, 3Fitzky B.U. Moebius F.F. Asaoka H. Waage-Baudet H. Xu L. Xu G. Maeda N. Kluckman K. Hiller S. 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Herein, we report 1) the isolation and characterization of five oxysterols from the brains of AY9944-treated rats, employing HPLC-ultraviolet (UV), HPLC-MS, and 1D- and 2D-NMR; 2) the HPLC-MS-MS oxysterol profiles of AY9944-treated and age-matched control rat brain, liver, and serum; 3) the chemical synthesis of two novel, 7-DHC-derived oxysterols (4α- and 4β-hydroxy-7-DHC); and 4) quantification of four of the five oxysterols in AY9944-treated and control rat brain, liver, and serum with HPLC-MS-MS using a deuterated standard of DHCEO. Unless stated otherwise, all biochemical and analytical reagents and solvents were of the highest purity, and used as obtained from various commercial vendors. Hexanes (HPLC grade), 2-propanol (HPLC grade), and other solvents were purchased from Thermo Fisher Scientific, Inc. Selenium dioxide, lead (IV) acetate, N-bromosuccinimide, benzoyl peroxide, trimethyl phosphite, and all other chemical reagents were purchased from Sigma-Aldrich Co., and were used without further purification. AY9944 [trans-1,4-bis(2-chlorobenzylaminomethyl) cyclohexane dihydrochloride] was prepared by custom organic synthesis (Dr. Abdul Fauq, Chemistry Core, Mayo Clinic, Jacksonville, FL) and was found to be identical in structure and purity (>99%) to an authentic sample of AY9944 previously obtained from Wyeth-Ayerst Laboratories, as determined by HPLC, 1H-NMR, and GC-MS. [25,26,26,26,27,27,27-d7]Cholesterol (99% D) was purchased from Medical Isotopes, Inc. [25,26,26,26,27,27,27-d7]7-DHC and [25,26,26,26,27,27,27-d7]DHCEO were synthesized as reported previously (32Xu L. Korade Z. Rosado D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2011; 52: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Sprague-Dawley rats were obtained from Harlan Laboratories, Inc. (Indianapolis, IN). The AY9944-induced SLOS rat model was generated as described in detail previously (41Fliesler S.J. Peachey N.S. Richards M.J. Nagel B.A. Vaughan D.K. Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.Arch. Ophthalmol. 2004; 122: 1190-1200Crossref PubMed Scopus (54) Google Scholar). All procedures were approved by the Institutional Animal Care and Use Committee of the Buffalo VA Medical Center and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All rats were maintained in dim cyclic light (20–40 lux, 12 h light/12 h dark) at 22–25°C and were provided cholesterol-free rodent chow (Purina Mills TestDiet, Richmond, IN) and water ad lib. At desired ages (2 to 3 months postnatal), rats were euthanized by sodium pentobarbital overdose, conforming to procedures approved by the American Veterinary Medical Association Panel of Euthanasia, and tissues were harvested, flash-frozen in liquid nitrogen, and stored in darkness at −80°C until ready for extraction and analysis of lipids (see below). Brain tissues (∼1.5 g) of 2 or 3 month-old rats were homogenized and extracted in a similar way as described previously (32Xu L. Korade Z. Rosado D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2011; 52: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). The organic layer was dried under nitrogen, reconstituted in methylene chloride, and subjected to separation on NH2-solid phase extraction cartridge [Phenomenex, 500 mg; solvents: condition with 4 ml hexane and elution with 4 ml chloroform/2-propanol (2:1) to collect neutral lipids including oxysterols]. The eluted fraction was divided into half, and each half was subjected to separation on normal-phase Si-SPE (1 g; solvents: condition with hexane, elution with 20 ml 0.5% 2-propanol in hexane to remove the majority of cholesterol and 7-DHC, elution with 10 ml 15% 2-propanol in hexane to collect the fraction that contains oxysterols). The resulting fractions containing oxysterols were subjected to separation with reverse-phase (RP)-HPLC-UV (150 × 2 mm C18 column; 3 μm; 0.2 ml/min; elution solvent: acetonitrile-methanol, 70:30, v/v), and thus obtained HPLC fractions were subsequently separated with normal-phase (NP)-HPLC-UV (Silica 4.6 mm × 25 cm column; 5 μ; 1.0 ml/min; elution solvent: 10% 2-propanol in hexane). Pure fractions were obtained in this way and were analyzed by one dimensional (1D)- and 2D-NMR spectroscopy to characterize the oxysterol structures as described in the text (also see Supplementary Materials). All samples were processed under dim red light. An appropriate amount of d7-DHCEO standard was added to each sample before sample processing. Brain tissues (vertical cut half brain) and liver tissues (∼100 mg cut off from the whole liver) were worked up similarly to what is described above, but without the Si-SPE procedure. Lipid extraction from serum (200 μl; each sample was spiked with 20.8 μg d7-7-DHC and 20.0 μg d7-cholesterol) was carried out in a similar procedure. Thus obtained organic layer from serum extraction was blown dry with nitrogen and was reconstituted in methanol (0.5 ml) and 1 M KOH in water (0.5 ml), and the resulting mixture was incubated at 37°C for 30 min. The hydrolyzed mixture was directly extracted with hexane (2 ml × 2), and the combined organic layers were dried under nitrogen, reconstituted in methylene chloride (200 μl), and stored at −80°C until analysis. Oxysterols in all samples were analyzed by NP-HPLC atmospheric pressure chemical ionization (APCI)-MS-MS following the previously reported method (32Xu L. Korade Z. Rosado D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2011; 52: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). NP-HPLC condition is the same as described above. For MS analysis, selective reaction monitoring (SRM) was employed to monitor the dehydration process of the ion [M+H]+ or [M+H-H2O]+ in the mass spectrometry (32Xu L. Korade Z. Rosado D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2011; 52: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Levels of cholesterol and 7-DHC were analyzed by GC for brain and liver using cholestanol as the external standard and by GC-MS for serum using d7-cholesterol and d7-7-DHC as the internal standard. GC and GC-MS conditions are the same as we previously reported (32Xu L. Korade Z. Rosado D.A. Liu W. Lamberson C.R. Porter N.A. An oxysterol biomarker for 7-dehydrocholesterol oxidation in cell/mouse models for Smith-Lemli-Opitz syndrome.J. Lipid Res. 2011; 52: 1222-1233Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Control serum was used in this test, and the work-up procedure was the same as described above unless otherwise noted. Appropriate amounts of oxysterols, including 4α- and 4β-hydroxy-7-DHC, 7-ketocholesterol (7-kChol), and DHCEO, were added to the lipid extracts before and after hydrolysis. d7-DHCEO standard was added to each sample after hydrolysis to quantify the remaining oxysterols. Samples were analyzed with NP-HPLC-MS-MS in the same way as described above. 4α- and 4β-Hydroxy-7-DHC and 7-kChol were found to be stable under the hydrolysis condition (all recovery rates were close to 100%). The recovery rate for DHCEO, however, was only 28%. For synthetic procedures and product characterization, see Supplementary Materials. Prior studies from our laboratory have indicated the presence of novel 7-DHC-derived oxysterols in brains of Dhcr7-null mice, but unambiguous identification and quantification of these presumed oxysterols was impaired by the relatively limited amount of available tissues, noncharacteristic mass spectra obtained from those compounds, and the lack of authentic oxysterol standards. The vertebrate brain is highly enriched in lipids (∼40–80%, by dry wt., depending on region), of which cholesterol constitutes ∼20% of the total (45Chavko M. Nemoto E.M. Melick J.A. Regional lipid composition in the rat brain.Mol. Chem. Neuropathol. 1993; 18: 123-131Crossref PubMed Scopus (40) Google Scholar). The levels of 7-DHC and cholesterol have been examined in brain and other tissues of AY9944-treated rats, in comparison with age- and sex-matched controls: typically, 7-DHC/cholesterol ratios are ≥4/1 in brain, retina, liver, and serum by one postnatal month of AY9944 treatment, and the ratio increases thereafter (e.g., >11/1 in liver and serum by three postnatal months) (22Fliesler S.J. Retinal degeneration in a rat model of Smith-Lemli-Opitz syndrome: thinking beyond cholesterol deficiency.Adv. Exp. Med. Biol. 2010; 664: 481-489Crossref PubMed Scopus (29) Google Scholar, 40Fliesler S.J. Richards M.J. Miller C. Peachey N.S. Marked alteration of sterol metabolism and composition without compromising retinal development or function.Invest. Ophthalmol. Vis. Sci. 1999; 40: 1792-1801PubMed Google Scholar, 41Fliesler S.J. Peachey N.S. Richards M.J. Nagel B.A. Vaughan D.K. Retinal degeneration in a rodent model of Smith-Lemli-Opitz syndrome: electrophysiologic, biochemical, and morphologic features.Arch. Ophthalmol. 2004; 122: 1190-1200Crossref PubMed Scopus (54) Google Scholar, 46Fliesler S.J. Bretillon L. The ins and outs of cholesterol in the vertebrate retina.J. Lipid Res. 2010; 51: 3399-3413Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Thus, brain tissues of 2 or 3 month-old AY9944-treated rats were used for oxysterol isolation to ensure substantial accumulation of 7-DHC and 7-DHC-derived oxysterols. Typically, lipids extracted from rat brain were separated on NH2-SPE and Si-SPE to give fractions that contained oxysterols, and these fractions were subjected to further separation on NP- or RP-HPLC-UV (see Materials and Methods). Typical chromatograms of NP- and RP-HPLC-UV separation are shown in Fig. 1 and newly identified oxysterols are denoted therein (vide infra). Pure fractions were isolated by combination of NP- and RP-HPLC-UVs illustrated above (see Materials and Methods), and their structures were elucidated by UV, MS, 1-dimensional (ID
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