Aglycon diversity of brain sterylglucosides: structure determination of cholesteryl- and sitosterylglucoside
2016; Elsevier BV; Volume: 57; Issue: 11 Linguagem: Inglês
10.1194/jlr.m071480
ISSN1539-7262
AutoresHisako Akiyama, Kazuki Nakajima, Yasuhiko Itoh, Tomoko Sayano, Yoko Ohashi, Yoshiki Yamaguchi, Peter Greimel, Yoshio Hirabayashi,
Tópico(s)Glycosylation and Glycoproteins Research
ResumoTo date, sterylglucosides have been reported to be present in various fungi, plants, and animals. In bacteria, such as Helicobacter pylori, proton NMR spectral analysis of isolated 1-O-cholesteryl-β-d-glucopyranoside (GlcChol) demonstrated the presence of an α-glucosidic linkage. By contrast, in animals, no detailed structural analysis of GlcChol has been reported, in part because animal-derived samples contain a high abundance of glucosylceramides (GlcCers)/galactosylceramides, which exhibit highly similar chromatographic behavior to GlcChol. A key step in vertebrate GlcChol biosynthesis is the transglucosylation reaction catalyzed by glucocerebrosidase (GBA)1 or GBA2, utilizing GlcCer as a glucose donor. These steps are expected to produce a β-glucosidic linkage. Impaired GBA1 and GBA2 function is associated with neurological disorders, such as cerebellar ataxia, spastic paraplegia, and Parkinson's disease. Utilizing a novel three-step chromatographic procedure, we prepared highly enriched GlcChol from embryonic chicken brain, allowing complete structural confirmation of the β-glucosidic linkage by 1H-NMR analysis. Unexpectedly, during purification, two additional sterylglucoside fractions were isolated. NMR and GC/MS analyses confirmed that the plant-type sitosterylglucoside in vertebrate brain is present throughout embryonic development. The aglycon structure of the remaining sterylglucoside (GSX-2) remains elusive due to its low abundance. Together, our results uncovered unexpected aglycon heterogeneity of sterylglucosides in vertebrate brain. To date, sterylglucosides have been reported to be present in various fungi, plants, and animals. In bacteria, such as Helicobacter pylori, proton NMR spectral analysis of isolated 1-O-cholesteryl-β-d-glucopyranoside (GlcChol) demonstrated the presence of an α-glucosidic linkage. By contrast, in animals, no detailed structural analysis of GlcChol has been reported, in part because animal-derived samples contain a high abundance of glucosylceramides (GlcCers)/galactosylceramides, which exhibit highly similar chromatographic behavior to GlcChol. A key step in vertebrate GlcChol biosynthesis is the transglucosylation reaction catalyzed by glucocerebrosidase (GBA)1 or GBA2, utilizing GlcCer as a glucose donor. These steps are expected to produce a β-glucosidic linkage. Impaired GBA1 and GBA2 function is associated with neurological disorders, such as cerebellar ataxia, spastic paraplegia, and Parkinson's disease. Utilizing a novel three-step chromatographic procedure, we prepared highly enriched GlcChol from embryonic chicken brain, allowing complete structural confirmation of the β-glucosidic linkage by 1H-NMR analysis. Unexpectedly, during purification, two additional sterylglucoside fractions were isolated. NMR and GC/MS analyses confirmed that the plant-type sitosterylglucoside in vertebrate brain is present throughout embryonic development. The aglycon structure of the remaining sterylglucoside (GSX-2) remains elusive due to its low abundance. Together, our results uncovered unexpected aglycon heterogeneity of sterylglucosides in vertebrate brain. Lipid glycosylation is a common feature in all three domains of life: bacteria, archaea, and eukaryotes. To date, sterol glucosylation has been encountered in bacteria, fungi, plants, and animals [see review (1.Grille S. Zaslawski A. Thiele S. Plat J. Warnecke D. The functions of steryl glycosides come to those who wait: recent advances in plants, fungi, bacteria and animals.Prog. Lipid Res. 2010; 49: 262-288Crossref PubMed Scopus (125) Google Scholar)], indicating its important role during the evolution of life. While bacteria, such as Helicobacter pylori, produce large amounts of 1-O-cholesteryl-α-d-glucopyranoside (2.Hirai Y. Haque M. Yoshida T. Yokota K. Yasuda T. Oguma K. Unique cholesteryl glucosides in Helicobacter pylori: composition and structural analysis.J. Bacteriol. 1995; 177: 5327-5333Crossref PubMed Scopus (159) Google Scholar), animals such as chickens and snakes (3.Wertz P.W. Stover P.M. Abraham W. Downing D.T. Lipids of chicken epidermis.J. Lipid Res. 1986; 27: 427-435Abstract Full Text PDF PubMed Google Scholar, 4.Abraham W. Wertz P.W. Burken R.R. Downing D.T. Glucosylsterol and acylglucosylsterol of snake epidermis: structure determination.J. Lipid Res. 1987; 28: 446-449Abstract Full Text PDF PubMed Google Scholar) produce 1-O-cholesteryl-β-d-glucopyranoside (GlcChol), also known as glucosyl-β-d-cholesterol. Recently, Marques et al. (5.Marques A.R. Mirzaian M. Akiyama H. Wisse P. Ferraz M.J. Gaspar P. Ghauharali-van der Vlugt K. Meijer R. Giraldo P. Alfonso P. et al.Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases.J. Lipid Res. 2016; 57: 451-463Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar) measured elevated levels of GlcChol not only in somal tissues derived from mouse models of Gaucher disease (GD) and Niemann-Pick disease type C, but also in the plasma of GD and Niemann-Pick disease type C patients. It is well-known that in GD, glucosylceramide (GlcCer) accumulates in the lysosomal compartment of macrophages, and that GD is also associated with a homozygous mutation in glucocerebrosidase (GBA)1 (6.Brady R.O. Kanfer J.N. Shapiro D. Metabolism of glucocerebrosides. ii. evidence of an enzymatic deficiency in Gaucher's disease.Biochem. Biophys. Res. Commun. 1965; 18: 221-225Crossref PubMed Scopus (585) Google Scholar, 7.Barranger J.A. Ginns E.I. Glucosylceramide lipidoses: Gaucher disease.in: Scriver C.R. Beaudet A.L. Sly W.S. The Metabolic Basis of Disease. McGraw-Hill, New York1989: 1677-1698Google Scholar, 8.Beutler E. Grabowski G.A. Gaucher disease.in: Scriver C.R. 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Interestingly, both enzymes also possess transferase activity, catalyzing the transfer of the glucose (Glc) residue from GlcCer to cholesterol (Chol) to yield GlcChol in mammalian cells (5.Marques A.R. Mirzaian M. Akiyama H. Wisse P. Ferraz M.J. Gaspar P. Ghauharali-van der Vlugt K. Meijer R. Giraldo P. Alfonso P. et al.Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases.J. Lipid Res. 2016; 57: 451-463Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar, 11.Akiyama H. Sasaki N. Hanazawa S. Gotoh M. Kobayashi S. Hirabayashi Y. Murakami-Murofushi K. Novel sterol glucosyltransferase in the animal tissue and cultured cells: evidence that glucosylceramide as glucose donor.Biochim. Biophys. Acta. 2011; 1811: 314-322Crossref PubMed Scopus (20) Google Scholar, 12.Akiyama H. Kobayashi S. Hirabayashi Y. Murakami-Murofushi K. Cholesterol glucosylation is catalyzed by transglucosylation reaction of β-glucosidase 1.Biochem. Biophys. Res. Commun. 2013; 441: 838-843Crossref PubMed Scopus (50) Google Scholar). As a consequence, GlcCer, a key intermediate in sphingolipid metabolism, is the precursor of GlcChol. Similar to GBA1, loss-of-function mutations in GBA2 are associated with neurological disorders, such as cerebellar ataxia and spastic paraplegia (13.Hammer M.B. Eleuch-Fayache G. Schottlaender L.V. Nehdi H. Gibbs J.R. Arepalli S.K. Chong S.B. Hernandez D.G. Sailer A. Liu G. et al.Mutations in GBA2 cause autosomal-recessive cerebellar ataxia with spasticity.Am. J. Hum. Genet. 2013; 92: 245-251Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 14.Martin E. Schule R. Smets K. Rastetter A. Boukhris A. Loureiro J.L. Gonzalez M.A. Mundwiller E. Deconinck T. Wessner M. et al.Loss of function of glucocerebrosidase GBA2 is responsible for motor neuron defects in hereditary spastic paraplegia.Am. J. Hum. Genet. 2013; 92: 238-244Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar, 15.Votsi C. Zamba-Papanicolaou E. Middleton L.T. Pantzaris M. Christodoulou K. A novel GBA2 gene missense mutation in spastic ataxia.Ann. Hum. Genet. 2014; 78: 13-22Crossref PubMed Scopus (34) Google Scholar). However, the exact molecular mechanism and specific contributions of GBA1 and GBA2 dysfunction to neuronal malfunction and degeneration are not fully elucidated. Nevertheless, the transferase activity of GBA1 and GBA2 represents an intriguing intersection between two major lipid metabolic pathways, namely sphingolipids and sterols. Consequently, sphingolipid-sterol cross-talk may be important in maintaining neuronal homeostasis, all in the context that its deregulation plays a crucial role in the pathogenesis of neurodegenerative disorders, such as GD and Parkinson's disease. While the presence of GlcChol in human and mouse tissue, such as brain, has been inferred by LC-ESI-MS/MS analyses (5.Marques A.R. Mirzaian M. Akiyama H. Wisse P. Ferraz M.J. Gaspar P. Ghauharali-van der Vlugt K. Meijer R. Giraldo P. Alfonso P. et al.Glucosylated cholesterol in mammalian cells and tissues: formation and degradation by multiple cellular beta-glucosidases.J. Lipid Res. 2016; 57: 451-463Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), its purification and complete structural analysis based on GlcChol-containing fractions are yet to be reported. Here, we describe the purification of the sterylglucoside fraction from embryonic chicken brain. Embryonic chicken brains were selected as starting material due to the well-investigated and comparatively discernable composition of their glycolipid fraction (16.Basu S. Schultz A.M. Basu M. Roseman S. Enzymatic synthesis of galactocerebroside by a galactosyltransferase from embryonic chicken brain.J. Biol. Chem. 1971; 246: 4272-4279Abstract Full Text PDF PubMed Google Scholar, 17.Basu S. Kaufman B. Roseman S. Enzymatic synthesis of glucocerebroside by a glucosyltransferase from embryonic chicken brain.J. Biol. Chem. 1973; 248: 1388-1394Abstract Full Text PDF PubMed Google Scholar). During development of the purification procedure, we placed special emphasis on removing the large excess of chromatographically similar galactosylceramide (GalCer) known to be present in the CNS of vertebrates. Structural analysis of the isolated sterylglucoside fraction revealed the presence of a variety of aglycons. In addition to the major component featuring the expected cholesteryl aglycon, at least two more steryl aglycons were encountered, including the plant-type sitosteryl. Our work described here is the first report to describe the complete structure of GlcChol and 1-O-sitosteryl-β-d-glucopyranoside [(GlcSito), also known as glucosyl-β-d-sitosterol] derived from vertebrate brain and to demonstrate that sterylglucosides have a heterogeneous aglycon composition. The GlcChol, GlcSito, and GalCer from bovine brain, deuterated chloroform (CDCl3; 99.96% D), and 2,5-dihydroxybenzoic acid were purchased from Sigma-Aldrich (St. Louis, MO). Cholesterol-d7, β-d-galactopyranosyl-(1→1)-N-lauroyl-d-erythro-sphingosine [GalCer (d18:1-C12:0)], and β-d-glucopyranosyl-(1→1)-N-steroyl- d-erythro-sphingosine [GlcCer (d18:1-C18:0)] were purchased from Avanti Polar Lipids (Alabaster, AL). The 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl 2,2,2-trichloroacetimidate was purchased from Tokyo Chemical Industry (Tokyo, Japan) and TMS was from Acros Organics (Geel, Belgium). Cerezyme®, a recombinant human GBA1 used in enzyme replacement therapy for GD (18.Grabowski G.A. Barton N.W. Pastores G. Dambrosia J.M. Banerjee T.K. McKee M.A. Parker C. Schiffmann R. Hill S.C. Brady R.O. Enzyme therapy in type 1 Gaucher disease: comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources.Ann. Intern. Med. 1995; 122: 33-39Crossref PubMed Scopus (412) Google Scholar), was purchased from Genzyme Japan (Tokyo, Japan). For LC-ESI-MS/MS, HPLC-grade acetonitrile and methanol were purchased from Thermo Fisher Scientific (Waltham, MA), chloroform and distilled water were from Kanto Chemical Co., Inc. (Tokyo, Japan), ammonium formate was from Sigma-Aldrich Japan (Tokyo, Japan), and ammonium acetate was from Wako (Osaka, Japan). Fertilized Boris Brown chicken eggs were purchased from Inoue Poultry Farm (Sagamihara, Japan) and maintained in a rocking egg incubator at 38°C. Upon reaching the appropriate stage, the embryos were sacrificed, either heads without eyes (6 days old) or brains (8–18 days old) were quickly harvested and immediately frozen in liquid nitrogen (N2). After lyophilization, samples were stored at −80°C until further use. Total lipids were extracted from lyophilized brains (290 brains, ∼7.0 g dry weight) of 10- to 12-day-old embryonic chickens using 200 ml of chloroform:methanol (C:M) (C:M at 2:1, v/v; C:M at 1:1, v/v; and C:M at 1:2, v/v). After evaporation, the combined extracts were hydrolyzed for 1 h at 37°C in C:M (2:1, v/v, 150 ml) containing 0.1 M KOH. The reaction mixture was subjected to Folch's partition, and the lower phase was evaporated to dryness. The resulting lipid film was resuspended in chloroform (50 ml) and applied to a column of silica gel 60 (Kanto Chemical Co.) equilibrated with chloroform. Glycolipids, including GlcChol, were eluted using a stepwise gradient, starting with pure chloroform (250 ml), and then with 250 ml each of C:M (98:2, v/v); C:M (95:5, v/v); C:M (9:1, v/v); and C:M (8:2, v/v). The fractions eluted with C:M (9:1, v/v) containing sterylglucoside alongside hexosylceramide were pooled and evaporated to dryness. The lipid film was resuspended in 6 ml of a methanol:water (M:W) mixture (M:W at 9:1, v/v) and subjected to reversed-phase (RP) column chromatography over silica gel 120 (RP-18; Kanto Chemical Co.), equilibrated with M:W (9:1, v/v). Elution was facilitated by using a stepwise gradient of M:W (95:5, v/v, 4 ml); M:W (98:2, v/v, 10 ml); and methanol (10 ml). The presence of sterylglucosides was evaluated using TLC, and positive fractions were pooled and dried. A portion of the enriched sterylglucoside fraction was further purified by RP-HPLC, as described previously (19.McDonald J.G. Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 145-170Crossref PubMed Scopus (125) Google Scholar) with minor modifications. Briefly, the lipid film was resuspended in a small volume of mobile phase B (M:W at 85:15, v/v), applied to an RP-HPLC Luna C18(2) column [4.6 mm inner diameter (i.d.) × 250 mm, particle size, 3 µm; Phenomenex, Torrance, CA], and eluted with the following gradient of mobile phase A (pure methanol): 2 min, 0%; 13 min, 0–100% linear gradient; 40 min, 100% (washing step); and 15 min, 0% (equilibration). The flow rate was kept constant at 0.6 ml/min, and the column was maintained at room temperature. Lipid detection with short wave UV light at 205 nm was disabled during elution to prevent potential damage of the UV-absorbing double bond at the C5-C6 position of the expected aglycon (see Fig. 2B). The eluent was manually collected into 14 fractions in the following volumes: fraction 1, 15 ml; fractions 2–13, 0.3–0.6 ml, and fraction 14, 22 ml. Subsequently, all fractions were subjected to LC-ESI-MS/MS analysis, utilizing multiple reaction monitoring (MRM). Aglycon release was effected by suspending a portion of the enriched sterylglucoside fraction in a total volume of 20 or 40 µl reaction buffer [50 mM citrate-phosphate buffer (pH 5.3), 0.25% Triton X-100, 0.6% sodium taurocholate, 1–2 µl of 100 µg/µl Cerezyme® in PBS] and incubation at 37°C for 16–20 h. The reaction was terminated by addition of 2 ml of C:M (2:1, v/v) and 460 or 480 µl of water [adjusted to a total volume of 500 µl (including the reaction mixture)] to facilitate lipid extraction after Folch's partition. The organic layer was separated, dried, and the sterol fraction was purified by TLC on silica gel 60 using hexane:diethyl ether:acetic acid (80:20:1, v/v/v) as an eluent. Lipids were stained by primuline reagent (0.01% primuline, 80% acetone) and visualized by long wave UV detection. The band comigrating with standard Chol (Wako, Osaka, Japan) was collected and extracted by Folch's partition. The organic layer containing the released aglycons was separated and dried under a stream of N2 gas. The lipid containing released aglycons was suspended in 25 µl of TMS at room temperature for 30 min. The resulting trimethylsilylated material was subjected to GC/MS analysis on a GCMS-QP2010 Ultra (Shimadzu, Kyoto, Japan) equipped with an Ultra1 capillary column (25 m × 0.2 mm, film thickness of 0.33 µm; Agilent Technologies Inc., Santa Clara, CA). We employed the following temperature gradient: from 180 to 250°C at a heating rate of 20°C/min and from 250 to 300°C at a heating rate of 5°C/min. The highly purified sterylglucoside fraction and authentic GlcChol standard were each dissolved in C:M (1:1, v/v) at a concentration of 1 µg/µl, mixed with MALDI matrix A [10 µg/µl of 2,5-dihydroxybenzoic acid in C:M (1:1, v/v)] and matrix B (1 µg/µl LiCl in water) at a ratio of 1:1:1 (v/v/v). From the resulting mixture, 0.6–1.5 µl were spotted onto a MTP 384-hole mirror finish stainless steel plate (JEOL Ltd., Tokyo, Japan) and dried. The samples were analyzed with a JMS-S3000 Spiral TOF (JEOL Ltd., Akishima, Japan) equipped with the TOF/TOF option (20.Satoh T. Sato T. Kubo A. Tamura J. Tandem time-of-flight mass spectrometer with high precursor ion selectivity employing spiral ion trajectory and improved offset parabolic reflectron.J. Am. Soc. Mass Spectrom. 2011; 22: 797-803Crossref PubMed Scopus (33) Google Scholar). A Nd-YLF laser pulse of 349 nm was operated at 250 Hz. For product-ion mass spectrum acquisition, helium collision gas was introduced. The collision energy was 20 keV to induce high-energy collision-induced dissociation (CID). LC-ESI-MS/MS analysis was performed on a semi-micro LC system 100XL (Eksigent, Dublin, CA) fused to a triple quadrupole linear ion trap mass spectrometer, QTRAP4500 (SCIEX, Toronto, Canada). The datasets were analyzed with MultiQuant and Analyst software (SCIEX). Target lipids were monitored in the MRM mode using specific precursor-product ion pairs, as detailed in Table 1. Ionization efficiency of GlcCer (d18:1-C12:0) and GalCer (d18:1-C12:0) were similar under the employed conditions.TABLE 1Analytical conditions used for the analysis by MRM methodsPrecursor Ion (Q1)Product Ion (Q3)Collision Energy (eV)Glucosylated sterolsGSX-1 (GlcChol)566.4aPrecursor ion (Q1) [M + NH4]+.369.4cProduct ion (Q3) aglycon-related ion.15GSX-2580.3aPrecursor ion (Q1) [M + NH4]+.383.3cProduct ion (Q3) aglycon-related ion.15GSX-3 (GlcSito)594.3aPrecursor ion (Q1) [M + NH4]+.397.3cProduct ion (Q3) aglycon-related ion.13GlcCersGlcCer (d18:1-C12:0)644.4bPrecursor ion (Q1) [M + H]+.264.2 or 464.4dProduct ion (Q3) long-chain base-related ions.43 or 21GlcCer (d18:1-C16:0)700.7bPrecursor ion (Q1) [M + H]+.264.2dProduct ion (Q3) long-chain base-related ions.45.5GlcCer (d18:1-C18:0)728.7bPrecursor ion (Q1) [M + H]+.264.2dProduct ion (Q3) long-chain base-related ions.48GlcCer (d18:1-C24:1)810.7bPrecursor ion (Q1) [M + H]+.264.2dProduct ion (Q3) long-chain base-related ions.55.5a Precursor ion (Q1) [M + NH4]+.b Precursor ion (Q1) [M + H]+.c Product ion (Q3) aglycon-related ion.d Product ion (Q3) long-chain base-related ions. Open table in a new tab Sterylglucosides were analyzed as described previously (19.McDonald J.G. Thompson B.M. McCrum E.C. Russell D.W. Extraction and analysis of sterols in biological matrices by high performance liquid chromatography electrospray ionization mass spectrometry.Methods Enzymol. 2007; 432: 145-170Crossref PubMed Scopus (125) Google Scholar), with minor modifications. Briefly, samples (∼200 µg dry weight) dissolved in 10 µl of C:M (2:1, v/v) were diluted 10-fold with mobile phase B [M:W at 85:15 (v/v), 5 mM ammonium acetate] and applied onto a RP column [Luna C18(2) column; 2 mm i.d. × 250 mm, particle size, 3 µm; Phenomenex] maintained at 36°C and at a flow rate of 0.15 ml/min. The samples were then eluted with the following gradients of mobile phase A (methanol pure, 5 mM ammonium acetate): 2 min, 0%; 13 min, 0–100% linear gradient; 40 min, 100% (washing step); 15 min 0% (equilibration). The mass spectrometer was set to positive ion mode (ion spray voltage, 5,500 V; curtain gas pressure, 20 psi; nebulizer gas pressure, 80 psi; heating gas pressure, 40 psi; temperature, 100°C), utilizing either MRM detection for targeted analysis or neutral-loss scan for untargeted analysis. The parameters for the latter were as follows: 197 Da, calculated as loss of hexose and NH3, as rationalized in supplemental Fig. S1; scan speed, 200 Da/s; collision energy, 15 eV. GlcCers were analyzed, as previously reported (21.Shaner R.L. Allegood J.C. Park H. Wang E. Kelly S. Haynes C.A. Sullards M.C. Merrill Jr, A.H. Quantitative analysis of sphingolipids for lipidomics using triple quadrupole and quadrupole linear ion trap mass spectrometers.J. Lipid Res. 2009; 50: 1692-1707Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 22.Nakajima K. Akiyama H. Tanaka K. Kohyama-Koganeya A. Greimel P. Hirabayashi Y. Separation and analysis of mono-glucosylated lipids in brain and skin by hydrophilic interaction chromatography based on carbohydrate and lipid moiety.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2016; 1031: 146-153Crossref PubMed Scopus (13) Google Scholar), by hydrophilic interaction chromatography (HILIC)-ESI-MS/MS with minor modifications. Concisely, samples (∼200 µg dry weight) dissolved in 10 µl of C:M (2:1, v/v) were diluted 10-fold with mobile phase A (acetonitrile:methanol:formic acid, 97:2:1, v/v/v, with 5 mM ammonium formate) and applied to an Atlantis silica HILIC column (2.1 mm i.d. × 150 mm, particle size, 3 µm; Waters, Milford, MA) maintained at 40°C. Samples were eluted at a flow rate of 0.15 ml/min, utilizing the following gradient of mobile phase B (methanol:water:formic acid, 89:9:1, v/v/v, with 20 mM ammonium formate): 3.3 min, 0%; 13.4 min, 0–35% linear gradient; 1.3 min, 35–70% linear gradient; 3 min, 70% (washing step); 30 min, 0%, flow rate increased to 0.2 ml/min (equilibration). The mass spectrometer was set to positive ion mode (ion spray voltage, 5,500 V; curtain gas pressure, 30 psi; nebulizer gas pressure, 90 psi; heating gas pressure, 30 psi; temperature, 100°C) utilizing MRM detection for targeted analysis. Highly purified sterylglucoside fractions and authentic standards were dissolved in CDCl3 containing tetramethylsilane as an internal chemical shift reference. One-dimensional 1H-NMR and two-dimensional double quantum filtered correlation spectroscopy (DQF-COSY) and homonuclear Hartmann-Hahn (HOHAHA) spectra, as well as 1H-13C multiplicity-edited heteronuclear single quantum coherence (HSQC) spectra were recorded on a DRX-500 spectrometer (Bruker BioSpin, Yokohama, Japan) equipped with a TXI cryogenic probe. Probe temperature was set at 25°C. The NMR data were processed with XWIN-NMR (version 3.5) and the spectra were displayed using XWIN-PLOT (version 3.5). The synthetic route to deuterium-labeled cholesteryl-β-d-glucoside (GlcChol-d7) is shown in supplemental Fig. S2. Unless stated otherwise, reactions were performed under argon. All solvents and chemicals were purchased as reagent grade from commercial suppliers and used without further purification, unless stated otherwise. Dry solvents were purchased from Kanto Chemical Co. and used as supplied. Analytical TLC and flash column chromatography were performed using the indicated solvent systems on Merck silica gel 60 F256 plates and on Kanto Chemical Co. silica gel 60 N (40–100 mesh), respectively. Low-resolution mass spectra (LRMS) were recorded on an SCIEX 4000 QTRAP mass spectrometer. NMR spectra were obtained on a JEOL ECA-500 spectrometer (1H at 500, 13C at 125 MHz) in the indicated solvents, with chemical shift referenced to residual nondeuterated solvent. Compound 3 (2,3,4,6-tetra-O-acetyl-1-O-cholesteryl-β-d-glucopyranoside; see supplemental Fig. S2) was synthesized as follows. Cholesterol-d7 (100 mg, 0.25 mmol) and 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl 2,2,2-trichloroacetimidate (150 mg, 0.3 mmol) were dissolved in dry dichloromethane (5 ml) at −40°C and stirred for 10 min. The reaction was initiated by addition of trimethylsilyl trifluoromethanesulfonate (5 µl) and stirred for 2 h at −40°C. Subsequently, the reaction was quenched with triethylamine (1 ml), and the volume was increased with dichloromethane (5 ml) prior to extraction against water and brine. The organic layer was dried over Na2SO4 and concentrated in vacuo. The residue was subjected to flash chromatography on silica gel (gradient of hexane:ethyl acetate at 10:1 to 1:1, v/v); product elution was monitored by TLC (hexane:ethyl acetate at 3:1, v/v), to give compound 3 as a white amorphous solid (112.5 mg, 0.16 mmol, 63% yield). 1H NMR (500 MHz, CDCl3, 25°C): δ = 5.35 (d, 1H, J = 5.2, H-6), 5.25 (dd, 1H, J = 9.2, J = 9.7, Glc H-3), 5.11 (dd, 1H, J = 9.7, J = 9.7, Glc H-4), 4.99 (dd, 1H, J = 9.7, J = 8, Glc H-2), 4.58 (d, 1H, J = 7.7, Glc H-1), 4.25 (dd, 1H, J = 12.3, J = 4.9, Glc H-6A), 4.10 (dd, 1H, J = 12, J = 2.3, Glc H-6B), 3.68 (n.r., 1H, Glc H-5), 3.48 (m, 1H, H-3), 2.25 (dd, 1H, J = 14.3, J = 4, Chol H-4A), 2.19 (dd, 1H, J = 11.2, J = 2, Chol H-4B), 2.07 (s, 3H, Ac), 2.04 (s, 3H, Ac), 2.01 (s, 3H, Ac), 2.00 (n.r., 1H, Chol H-12A), 2.00 (s, 3H, Ac), 1.96 (n.r., 1H, Chol H-7A), 1.89 (n.r., 1H, Chol H-2A), 1.85 (n.r., 1H, Chol H-1A), 1.82 (n.r., 1H, Chol H-16A), 1.58 (n.r., 1H, Chol H-2B), 1.55 (n.r., 1H, Chol H-15A), 1.48 (n.r., 1H, Chol H-7B), 1.46 (n.r., 1H, Chol H-11A), 1.42 (n.r., 1H, Chol H-8), 1.40 (n.r., 1H, Chol H-20), 1.40 (n.r., 1H, Chol H-11B), 1.32 (n.r., 1H, Chol H-23A), 1.31 (n.r., 1H, Chol H-22A), 1.26 (n.r., 1H, Chol H-16B), 1.14 (n.r., 1H, Chol H-23B), 1.14 (n.r., 1H, Chol H-12B), 1.09 (n.r., 2H, Chol H-24), 1.08 (n.r., 1H, Chol H-15B), 1.07 (n.r., 1H, Chol H-17), 1.02 (n.r., 1H, Chol H-1B), 0.99 (n.r., 3H, Chol H-22B), 0.97 (n.r., 1H, Chol H-19), 0.94 (n.r., 3H, Chol H-14), 0.90 (n.r., 1H, Chol H-21), 0.90 (n.r., 1H, Chol H-9), 0.67 (n.r., 3H, Chol H-18); 13C NMR (125 MHz, CDCl3, 25°C): δ = 170.8, 170.5, 169.5, 169.4 (4C, Ac), 140.4 (1C, Chol C-5), 122.3 (1C, Chol C-6), 99.7 (1C, Glc C-1), 80.2 (1C, Chol C-3), 73 (1C, Glc C-3), 71.9 (1C, Glc C-5), 71.6 (1C, Glc C-2), 68.6 (1C, Glc C-4), 62.2 (1C, Glc C-6), 56.8 (1C, Chol C-14), 56.2 (1C, Chol C-17), 50.3 (1C, Chol C-9), 42.4 (1C, Chol C-13), 39.8 (1C, Chol C-12), 39.3 (1C, Chol C-24), 39 (1C, Chol C-4), 37.3 (1C, Chol C-1), 36.8 (1C, Chol C-10), 36.3 (1C, Chol C-22), 35.9 (1C, Chol C-20), 32 (1C, Chol C-7), 32 (1C, Chol C-8), 29.5 (1C, Chol C-2), 28.3 (1C, Chol C-16), 24.4 (1C, Chol C-15), 23.9 (1C, Chol C-23), 21.1 (1C, Chol C-11), 20.9, 20.8, 20.7, 20.7 (4C, Ac), 19.4 (1C, Chol C-19), 18.8 (1C, Chol C-21), 11.9 (1C, Chol C-18); LRMS (ESI, pos) calcd. for C41H57D7O10 [M + Na]+: 746.48, found: 746.5. Compound 4 (1-O-cholesteryl-β-d-glucopyranoside; see supplemental Fig. S2) was synthesized as follows. Dry methanol (9 ml) and dry dioxane (1 ml) were mixed and treated with sodium (∼5 mg). After the initial reaction subsided and the mixture cooled to room temperature, it was transferred under Schlenk conditions to a new reaction vessel charged with compound 3 (100 mg, 0.14 mmol). Reaction progress was monitored by TLC (C:M at 9:1, v/v) and, after 2 h stirring at room temperature, the reaction mixture was dried in vacuo. The residue was subjected to flash chromatography on silica gel (gradient of C:M at 50:1 to 5:1, v/v) to yield compound 4 as a white amorphous solid (33 mg, 0.06 mmol, 43% yield). Finally, a portion was subjected to RP-HPLC purification as detailed above (see Isolation of sterylglucosides) prior to its application as an internal standard for MS-based sterylglucoside quantification. 1H NMR (500 MHz, CDCl3, 25°C): δ = 5.33 (n.r., 1H, Chol H-6), 4.36 (d, 1H, J = 7.4, Glc H-1), 3.82 (dd, 1H, J = 12.2, J = 1.3, Glc H-6A), 3.66 (dd, 1H, J = 12, J = 5.2, Glc H-6B), 3.56 (m, 1H, Chol H-3), 3.34 (dd, 1H, J = 8.6, J = 8.6, Glc H-3), 3.33 (dd, 1H, J = 8.6, J = 6.9, Glc H-4), 3.24 (m, 1H, Glc H-5), 3.15 (dd, 1H, J = 8.6, J = 8, Glc H-2), 2.38 (dd, 1H, J = 12.9, J = 2.6, Chol H-4A), 2.23 (dd, 1H, J = 12, J = 12, Chol H-4B), 2.00 (n.r., 1H, Chol H-12A), 1.94 (n.r., 1H, Chol H-7A), 1.90 (n.r., 1H, Chol H-2A), 1.84 (n.r., 1H, Chol H-12A), 1.82 (n.r., 1H, Chol H-16A), 1.58 (n.r., 1H, Chol H-2B), 1.55 (n.r., 2H, Chol H-15), 1.48 (n.r., 1H, Chol H-11A), 1.47 (n.r., 1H, Chol H-7B), 1.42 (n.r., 1H, Chol H-11B), 1.41 (n.r., 1H, Chol H-8), 1.38 (n.r., 1H, Chol H-20), 1.31 (n.r., 2H, Chol H-23), 1.30 (n.r., 1H, Chol H-22A), 1.25 (n.r., 1H, Chol H-16B), 1.14 (n.r., 1H, Chol H-12B), 1.08 (n.r., 2H, Chol H-24), 1.07 (n.r., 1H, Chol H-17), 1.06 (n.r., 1H, Chol H-1B), 0.99 (n.r., 3H, Chol H-19), 0.97 (n.r., 1H, Chol H-14), 0.97 (n.r., 1H, Chol H-22B), 0.90 (n.r., 1H, Chol H-9), 0.89 (n.r., 3H, Chol H-21), 0.66 (n.r., 3H, Chol H-18); 13C NMR (125 MHz, C
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