Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?
2009; Elsevier BV; Volume: 285; Issue: 7 Linguagem: Inglês
10.1074/jbc.m109.086678
ISSN1083-351X
AutoresMichael Ogundare, Spyridon Theofilopoulos, Andrew Lockhart, Leslie J. Hall, Ernest Arenas, Jan Sjövall, A.G. Brenton, Yuqin Wang, William J. Griffiths,
Tópico(s)Peroxisome Proliferator-Activated Receptors
ResumoIn this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease. In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C27 and C24 intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C26 sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ5-C27-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease. IntroductionThe steroid profile of the central nervous system is of considerable interest with respect to neurodegenerative disease (1Björkhem I. Cedazo-Minguez A. Leoni V. Meaney S. Mol. Aspects Med. 2009; 30: 171-179Crossref PubMed Scopus (207) Google Scholar, 2Griffiths W.J. Wang Y. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 2778-2805Crossref PubMed Scopus (46) Google Scholar, 3Leoni V. Scand. J. Clin. Lab. Invest. 2009; 69: 22-25Crossref PubMed Scopus (58) Google Scholar). This is partly because of the high levels of cholesterol (cholest-5-en-3β-ol) in the central nervous system (4Lütjohann D. Acta Neurol. Scand. 2006; 185: 33-42Crossref Scopus (49) Google Scholar, 5Dietschy J.M. Turley S.D. J. Lipid Res. 2004; 45: 1375-1397Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar), the potential neuroprotective role of neurosteroids (6Weill-Engerer S. David J.P. Sazdovitch V. Liere P. Eychenne B. Pianos A. Schumacher M. Delacourte A. Baulieu E.E. Akwa Y. J. Clin. Endocrinol. Metab. 2002; 87: 5138-5143Crossref PubMed Scopus (283) Google Scholar), and the ability of some cholesterol metabolites to act as ligands to nuclear receptors, which are themselves implicated in neurodegenerative disease, e.g. the liver X receptors (LXRs) 6The abbreviations used are: LXRliver X receptorCSFcerebrospinal fluidCYPcytochrome P450FXRfarnesoid X receptorGPGirard PHSDhydroxysteroid dehydrogenaseISinternal standardLC-MSliquid chromatography-mass spectrometryLITlinear ion trapMRMmultiple reaction monitoringMSnmass spectrometry with multiple fragmentationNURR1nuclear receptor related 1RICreconstructed ion chromatogramRXRretinoid X receptorSPEsolid phase extractionHPLChigh pressure liquid chromatography. in Alzheimer disease (7Fan J. Donkin J. Wellington C. Biofactors. 2009; 35: 239-248Crossref PubMed Scopus (83) Google Scholar). Furthermore, brain-derived cholesterol metabolites represent biomarkers for cerebral cholesterol homeostasis, which is deranged in certain neurodegenerative disease, and thus their measurement in body fluids offers a marker for the progression of such disorders (8Björkhem I. J. Intern. Med. 2006; 260: 493-508Crossref PubMed Scopus (272) Google Scholar). In this regard, urine and blood represent the most accessible body fluids (9Alvelius G. Hjalmarson O. Griffiths W.J. Björkhem I. Sjövall J. J. Lipid Res. 2001; 42: 1571-1577Abstract Full Text Full Text PDF PubMed Google Scholar, 10Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjövall J. Wang Y. J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar), but their composition is highly dependent on the activity of other organs. Alternatively, cerebrospinal fluid (CSF), although being less readily accessible, bathes the central nervous system, and its content is more likely to reflect cholesterol metabolism in brain itself. With this in mind, we have set to profile the sterol content of human CSF.The levels of sterols in CSF are comparatively low; even cholesterol is present at a level of only 4–5 μg/ml (cf. 2 mg/ml in plasma) (11Leoni V. Lütjohann D. Masterman T. J. Lipid Res. 2005; 46: 191-195Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 12Schönknecht P. Lütjohann D. Pantel J. Bardenheuer H. Hartmann T. von Bergmann K. Beyreuther K. Schröder J. Neurosci. Lett. 2002; 324: 83-85Crossref PubMed Scopus (134) Google Scholar), whereas the brain-derived oxysterol 24(S)-hydroxycholesterol (cholest-5-ene-3β,24(S)-diol, C5-3β,24(S)-diol) is present at a level of only about 1.5 ng/ml (cf. 40–60 ng/ml in plasma) (supplemental Table S1) (11Leoni V. Lütjohann D. Masterman T. J. 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It should be noted that sulfated and glucuronidase oxysterols have also been found in plasma (15Yang Y. Griffiths W.J. Nazer H. Sjövall J. Biomed. Chromatogr. 1997; 11: 240-255Crossref PubMed Scopus (48) Google Scholar, 16Meng L.J. Griffiths W.J. Nazer H. Yang Y. Sjövall J. J. Lipid Res. 1997; 38: 926-934Abstract Full Text PDF PubMed Google Scholar) and that there is the possibility that sulfated sterols also have biological activity (17Fine J.M. Sorensen P.W. J. Chem. Ecol. 2008; 34: 1259-1267Crossref PubMed Scopus (52) Google Scholar). 24(S)-Hydroxycholesterol is a net export product from the central nervous system (18Lütjohann D. Breuer O. Ahlborg G. Nennesmo I. Sidén A. Diczfalusy U. Björkhem I. Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 9799-9804Crossref PubMed Scopus (561) Google Scholar) and is ultimately transported to the liver where it is metabolized into bile acids (19Russell D.W. Annu. Rev. Biochem. 2003; 72: 137-174Crossref PubMed Scopus (1347) Google Scholar, 20Björkhem I. Andersson U. Ellis E. Alvelius G. Ellegard L. Diczfalusy U. Sjövall J. Einarsson C. J. Biol. Chem. 2001; 276: 37004-37010Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). On the other hand, 27-hydroxycholesterol (cholest-5-ene-3β,26-diol, C5-3β,26-diol) is a net import product to the central nervous system (21Heverin M. Meaney S. Lütjohann D. Diczfalusy U. Wahren J. Björkhem I. J. Lipid Res. 2005; 46: 1047-1052Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar), and recent data suggest that it is metabolized in brain to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (CA4-7α-ol-3-one) (22Meaney S. Heverin M. Panzenboeck U. Ekström L. Axelsson M. Andersson U. Diczfalusy U. Pikuleva I. Wahren J. Sattler W. Björkhem I. J. Lipid Res. 2007; 48: 944-951Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). This acid is an intermediate in the acidic bile acid biosynthetic pathway (19Russell D.W. Annu. Rev. 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Lipid Res. 2004; 45: 1741-1748Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar) demonstrated the conversion of 3β-hydroxychol-5-en-24-oic acid (BA5-3β-ol) to chenodeoxycholic acid (3α,7α-dihydroxy-5β-cholan-24-oic acid, 5β-BA-3α,7α-diol) via 3β,7α-dihydroxychol-5-en-24- oic acid (BA5-3β,7α-diol) and 7α-hydroxy-3-oxochol-4-en-24-oic acid (BA4-7α-ol-3-one) in rat brain tissue. Mano et al. (26Mano N. Goto T. Uchida M. Nishimura K. Ando M. Kobayashi N. Goto J. J. Lipid Res. 2004; 45: 295-300Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) also demonstrated the presence of chenodeoxycholic acid, deoxycholic acid (3α,12α-dihydroxy-5β-cholan-24-oic acid, 5β-BA-3α,12α-diol), and cholic acid (3α,7α,12α-trihydroxy-5β-cholan-24-oic acid, 5β-BA-3α,7α,12α-triol) in rat brain, the chenodeoxycholic acid level being about 30 times greater than in serum. Furthermore, both C24 and C27 bile acids have been identified in human brain (27Ferdinandusse S. Denis S. Faust P.L. Wanders R.J. J. Lipid Res. 2009; 50: 2139-2147Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Despite their presence in brain and blood (28Axelson M. Mörk B. Sjövall J. J. Lipid Res. 1988; 29: 629-641Abstract Full Text PDF PubMed Google Scholar), there are few reports of the presence of bile acids and their precursors in CSF of healthy individuals, although 7α-hydroxy-3-oxocholest-4-en-26-oic acid has been found in the CSF of individuals who underwent surgery for aneurysmal subarachnoid hemorrhage (29Nagata K. Seyama Y. Shimizu T. Neurol. Med. Chir. 1995; 35: 294-297Crossref PubMed Scopus (5) Google Scholar). The same group also identified high concentrations of this acid in chronic subdural hematoma (30Nagata K. Takakura K. Asano T. Seyama Y. Hirota H. Shigematsu N. Shima I. Kasama T. Shimizu T. Biochim. Biophys. Acta. 1992; 1126: 229-236Crossref PubMed Scopus (17) Google Scholar).Sterols and bile acids have traditionally been analyzed by gas chromatography-mass spectrometry; however, liquid chromatography (LC)-MS and LC-tandem mass spectrometry (tandem mass spectrometry or MSn) offers an attractive alternative (31Griffiths W.J. Sjövall J. J. Lipid Res. 2010; 51: 23-41Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In this study, we have chosen to focus our attention on cholesterol metabolites, which are intermediates in the bile acid biosynthetic pathways, and to pay particular interest to those that possess either a 3β-hydroxy-5-ene or 3-oxo-4-ene structure in the AB rings (the ultimate primary bile acids have a 3α,7α-dihydroxy-5β(H) structure). To improve the response of such metabolites in LC-MS analysis when utilizing electrospray ionization, we have utilized a charge-tagging approach where analyte molecules are specifically tagged with a charged group to enhance their mass spectrometric detection (10Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjövall J. Wang Y. J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar, 32Griffiths W.J. Wang Y. Alvelius G. Liu S. Bodin K. Sjövall J. J. Am. Soc. Mass Spectrom. 2006; 17: 341-362Crossref PubMed Scopus (95) Google Scholar, 33Karu K. Hornshaw M. Woffendin G. Bodin K. Hamberg M. Alvelius G. Sjövall J. Turton J. Wang Y. Griffiths W.J. J. Lipid Res. 2007; 48: 976-987Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar, 34Wang Y. So K.M. Bodin K. Theofilopoulos S. Sacchetti P. Hornshaw M. Woffendin G. Karu K. Sjövall J. Arenas E. Griffiths W.J. Mol. 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For instance, 3-oxocholest-4-en-26-oic acid (rechristened Δ4-dafachronic acid) is a ligand for an orphan nuclear receptor (DAF12) in the nematode Caenorhabditis elegans (39Motola D.L. Cummins C.L. Rottiers V. Sharma K.K. Li T. Li Y. Suino-Powell K. Xu H.E. Auchus R.J. Antebi A. Mangelsdorf D.J. Cell. 2006; 124: 1209-1223Abstract Full Text Full Text PDF PubMed Scopus (358) Google Scholar), and 3β-hydroxycholest-5-en-26-oic acid has been shown to activate LXRα in human embryonic kidney 293 cells (40Song C. Liao S. Endocrinology. 2000; 141: 4180-4184Crossref PubMed Google Scholar). To clarify the question of biological function, we have tested the biological activity of a number of bile acids identified here in CSF as ligands for the LXRs α and β, both of which are expressed in brain (41Sacchetti P. So K.M. Hall A.C. Liste I. Steffensen K.R. Theofilopoulos S. Parish C.L. Hazenberg C. Richter L.A. Hovatta O. Gustafsson J.A. Arenas E. 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Each of these nuclear receptors form obligate heterodimers with the retinoid X receptor (RXR) and regulate gene expression through binding to response elements in the promoter regions of target genes.EXPERIMENTAL PROCEDURESMaterialsHPLC water and HPLC grade solvents were from Fisher or Sigma. Authentic sterols, bile acids, and their precursors were from Avanti Polar Lipids (Alabaster, AL), Steraloids Inc. (London, UK), Sigma, or from previous studies in our laboratories (10Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjövall J. Wang Y. J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar, 32Griffiths W.J. Wang Y. Alvelius G. Liu S. Bodin K. Sjövall J. J. Am. Soc. Mass Spectrom. 2006; 17: 341-362Crossref PubMed Scopus (95) Google Scholar, 34Wang Y. So K.M. Bodin K. Theofilopoulos S. Sacchetti P. Hornshaw M. Woffendin G. Karu K. Sjövall J. Arenas E. Griffiths W.J. Mol. Biosyst. 2009; 5: 529-541Crossref PubMed Scopus (31) Google Scholar). GP reagent (1-(carboxymethyl)pyridinium chloride hydrazide) was from TCI Europe (Oxford, UK), and cholesterol oxidase from Streptomyces sp. was from Sigma. Sep-Pak tC18 200-mg cartridges were from Waters. Luer-lock syringes were from BD Biosciences. CSF samples from nine subjects were part of a GlaxoSmithKline study and were provided with institutional review board and ethical approval.MethodsExtraction of CSF for Analysis of SterolsCSF (0.5 ml) was added dropwise to 2.1 ml of 99.9% ethanol, containing 10 μl of 24(RS)-[26,26,26,27,27,27-2H6]hydroxycholesterol (Avanti Polar Lipids) in propan-2-ol (4 ng/μl), in an ultrasonic bath. This solution was diluted to 70% ethanol by the addition of 0.4 ml of water, ultrasonicated for 2 min, and centrifuged at 14,000 × g at 4 °C for 30 min.A 200-mg Sep-Pak tC18 cartridge (SPE1) was rinsed with 4 ml of 99.9% ethanol followed by 6 ml of 70% ethanol. CSF in 70% ethanol (3 ml) was applied to the cartridge and allowed to flow at a rate of ∼0.25 ml/min, and flow was aided by application of a slight pressure from a Luer-lock syringe. The flow-through and a column wash of 4 ml of 70% ethanol were collected (SPE1-Fr-1, Scheme 1). By testing the method with a solution of cholesterol and 25-hydroxycholesterol (cholest-5-ene-3β,25-diol, C5-3β,25-diol) in 70% ethanol, cholesterol was found to be retained on the column even after a 4-ml column wash, whereas 25-hydroxycholesterol elutes in the flow-through and column wash. Following a further wash with 4 ml of 70% ethanol (SPE1-Fr-2), cholesterol was eluted from the Sep-Pak column with 2 ml of 99.9% ethanol (SPE1-Fr-3). The column can be further stripped with an additional 2-ml aliquot of 99.9% ethanol to elute more hydrophobic sterols (SPE1-Fr-4). Each fraction was dried under reduced pressure using a vacuum concentrator (ScanLaf, Denmark).Charge Tagging of SterolsThe sterol fractions from above were reconstituted in 100 μl of propan-2-ol, and a solution of 1 ml of 50 mm phosphate buffer (KH2PO4, pH 7) containing 3.0 μl of cholesterol oxidase (2 mg/ml in H2O, 44 units/mg of protein) was added to each. The mixture was incubated at 37 °C for 1 h and then quenched with 2 ml of methanol (Scheme 1, route B, and Scheme 2).SCHEME 2Charge tagging of sterols and bile acids as exemplified by 3β-hydroxycholest-5-en-26-oic acid.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Glacial acetic acid (150 μl) was added to the reaction mixture above (now in ∼70% methanol), followed by 150 mg of GP reagent. The mixture was thoroughly vortexed and incubated at room temperature overnight in the dark.SPE Extraction of Charge-tagged SterolsEven when derivatized with GP reagent, sterols may be difficult to solubilize (or retain in solution) when using a highly aqueous mixture of methanol and water. This can make their extraction using reversed phase-solid phase extraction (SPE) challenging. To circumvent this problem a recycling procedure is used (10Griffiths W.J. Hornshaw M. Woffendin G. Baker S.F. Lockhart A. Heidelberger S. Gustafsson M. Sjövall J. Wang Y. J. Proteome Res. 2008; 7: 3602-3612Crossref PubMed Scopus (63) Google Scholar, 32Griffiths W.J. Wang Y. Alvelius G. Liu S. Bodin K. Sjövall J. J. Am. Soc. Mass Spectrom. 2006; 17: 341-362Crossref PubMed Scopus (95) Google Scholar).A 200-mg Sep-Pak tC18 cartridge (SPE2) was washed with 6 ml of 100% methanol, 6 ml of 10% methanol and conditioned with 4 ml of 70% methanol. The derivatization mixture from above (∼3 ml of 70% methanol, 5% acetic acid, 3% propanol-2-ol, containing 150 mg of GP reagent and 6 μg of cholesterol oxidase) was applied to the column followed by 1 ml of 70% methanol and 1 ml of 35% methanol. The combined effluent (5 ml) was diluted with water (4 ml) to give 9 ml of ∼35% methanol. The resulting solution was again applied to the column followed by a wash of 1 ml of 17% methanol. To the combined effluent, 9 ml of water was added to give 19 ml of ∼17.5% methanol. This solution was again applied to the column followed by a wash with 6 ml of 10% methanol. At this point, all the derivatized sterols were extracted by the column, and excess derivatization reagent was in the flow-through and wash. Derivatized sterols were then eluted in three 1-ml portions of 100% methanol (SPE2-Fr-1, Fr-2, and Fr-3) followed by 1 ml of 99.9% ethanol (SPE2-Fr-4). LC-MSn analysis revealed that the derivatized sterols were present almost exclusively in the first 2 ml of methanol eluent (SPE2-Fr-1 and SPE2-Fr-2). The recovery of 24(RS)-[26,26,26,27,27,27-2H6]hydroxycholesterol was estimated to be in excess of 85%.Cholesterol oxidase converts sterols with a 3β-hydroxy-5-ene function to 3-oxo-4-ene analogues (Scheme 2) and a 3β-hydroxy-5α-hydrogen function to a 3-oxo function. To identify sterols that naturally possess a 3-oxo function from those oxidized to contain one, CSF samples were analyzed in parallel in the presence (Scheme 1, route B) and absence (Scheme 1, route A) of cholesterol oxidase.LC-MSn on the LTQ-Orbitrap XLChromatographic separation of GP-tagged sterols was performed on an Ultimate 3000 HPLC system (Dionex, Surrey, UK) utilizing a Hypersil GOLD reversed phase column (1.9 μm particles, 50 × 2.1 mm, Thermo Fisher, San Jose, CA). Mobile phase A consisted of 33.3% methanol, 16.7% acetonitrile containing 0.1% formic acid, and mobile phase B consisted of 63.3% methanol 31.7% acetonitrile containing 0.1% formic acid. After 1 min at 20% B, the proportion of B was raised to 80% B over the next 7 min and maintained at 80% B for a further 5 min, before returning to 20% B in 6 s and re-equilibration for a further 3 min, 54 s, giving a total run time of 17 min. The flow rate was maintained at 200 μl/min and eluent directed to the atmospheric pressure ionization source of an LTQ-Orbitrap XL (Thermo Fisher, San Jose, CA) mass spectrometer. This instrument is a hybrid linear ion-trap (LIT)-Orbitrap analyzer. The Orbitrap is a Fourier transform mass analyzer capable of high resolution (up to 100,000 full width at half-maximum height) and exact mass measurement.The Orbitrap was calibrated externally prior to each analytical session. Mass accuracy was better than 5 ppm. In any given chromatographic run, and in the mass range of GP-tagged sterols, measured mass values were found to be offset from the theoretical mass by a constant value ranging from +1 to +2 millimass units, for example. For LC-MS and LC-MSn analysis of reference compounds, the sample (1 pg/μl in 60% methanol, 0.1% formic acid) was injected (20 μl) onto the reversed phase column and eluted into the LTQ-Orbitrap at a flow rate of 200 μl/min. Two experimental methods were utilized. In the first experimental method, three scan events were performed as follows: a Fourier transform-MS scan in the Orbitrap analyzer over the m/z range 400–650 (or 300–800) at 30,000 resolution (full width at half-maximum height) with a maximum ion fill time of 500 ms, followed by data-dependent MS2 and MS3 events performed in the LIT with maximum ion fill times of 200 ms. For the MS2 and MS3 scans, three microscans were performed, the precursor ion isolation width was set at 2 (to select the monoisotopic ion) and the normalized collision energy at 30 and 35 (instrument settings), respectively. A precursor ion inclusion list was defined according to the m/z of the [M]+ ions of expected sterols (see supplemental Table S2) so that MS2 was preferentially performed on these ions in the LIT if their intensity exceeded a pre-set minimum (500 counts). If a fragment ion corresponding to a neutral loss of 79 Da from the precursor ion was observed in the MS2 event and was above a minimal signal setting (200 counts), MS3 was performed on this fragment. To maximize efficiency, the MS2 and MS3 event was performed at the same time as the high resolution mass spectrum was being recorded in the Orbitrap. The second experimental method involved a targeted multiple reaction monitoring approach (MRM). In event 1, the Orbitrap analyzer was scanned as above, and in event 2, the transition 534.4→455.4→ was monitored using collision energies of 30 and 35 for the MS2 and MS3 events, respectively (Scheme 3a). In event 3, the transition 540.4→461.4→ was monitored in a similar manner (to accommodate the 24(RS)-[2H6]hydroxycholesterol internal standard).SCHEME 3Fragmentation of GP-tagged sterols and bile acids. a, major MS2 fragmentation routes for GP-tagged sterols and bile acids exemplified by 27-hydroxycholesterol. A 3-oxo-4-ene functionality was generated by oxidation of the native 3β-hydroxy-5-ene function by cholesterol oxidase prior to treatment with GP reagent. b and c, structurally informative fragment ions observed in MS3 ([M]+→[M − 79]+→) spectra of GP-tagged sterols exemplified by 27-hydroxycholesterol following cholesterol oxidase treatment (b) and 7α-hydroxycholesterol after similar treatment (c).View Large Image Figure ViewerDownload Hi-res image Download (PPT)For the analysis of GP-tagged sterols from CSF, 12 μl of the first methanol fraction (1 ml) from the second SepPak C18 cartridge (SPE2-Fr-1) (equivalent to 6 μl of CSF assuming all the sterols elute in this methanol fraction) was diluted with 8 μl of 0.1% formic acid and 20 μl injected onto the LC column. MS, MS2, and MS3 spectra were recorded as described above. Other fractions from the SPE columns were analyzed in an identical fashion.Quantification and Isotope Dilution Mass SpectrometryThe quantities of identified sterols and bile acids in CSF were determined by isotope dilution mass spectrometry against a known amount of added 24(SR)-[2H6]hydroxycholesterol (100% [2H6]) internal standard (IS). For monohydroxycholesterols (C5-3β,x-diol), which are present in CSF in their free form in low amounts (<1 ng/ml), quantification was performed using the MRM transitions 534.4→455.4→ for the GP-tagged sterols and 540.4→461.4→ for GP-tagged 24(SR)-[2H6]hydroxycholesterol IS. Peak areas were used for calculation of concentration (see Equation 1). Bile acids were present in greater abundance than monohydroxycholesterols, and this allowed their quantification from reconstructed ion chromatograms (RICs) recorded on the Orbitrap. RICs were generated from spectra recorded at 30,000 resolution, with an m/z tolerance of 10 ppm. Again peak areas were determined, and analyte levels were calculated by applying Equation 1. Analyte peak area/IS peak area=analyte concentration/IS concentration(Eq. 1) Equation 1 assumes that all analytes have an identical response factor to the internal standard, which is true for 3β-hydroxy-5-ene and 3-oxo-4-ene sterols without additional substituents in the A-ring (33Karu K. Hornshaw M. Woffendin G. Bodin K. Hamberg M. Alvelius G. Sjövall J. Turton J. Wang Y. Griffiths W.J. J. Lipid Res. 2007; 48: 976-987Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In some experiments, the IS was not included, in which case relative abundances (%RA) were determined against 7α-hydroxy-3-oxocholest-4-en-26-oic acid, the most abundant sterol/bile acid component found in CSF, by applying Equation 2. (Analyte peak area/CA4−7α−ol−3−one peak area)×100%=%RA(Eq. 2) Luciferase Reporter AssayThe ability of the acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic, 3β,7α-dihydroxycholest-5-e
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