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• W2151025672 abstract "High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r2 = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with 2H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of 2H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols. High resolution LC/MS-MS and LC/APPI-MS methods have been established for the quantitation of flux in the turnover of cholesterol and cholesterol ester. Attention was directed toward quantifying the monoisotopic mass (M0) and that of the singly deuterated labeled (M+1) isotope. A good degree of isotopic dynamic range has been achieved by LC/MS-MS ranging from 3-4 orders of magnitude. Correlation between the linearity of GC/MS and LC atmospheric pressure photoionization (APPI)-MS are complimentary (r2 = 0.9409). To prove the viability of this particular approach, male C57Bl/6 mice on either a high carbohydrate (HC) or a high fat (HF) diet were treated with 2H2O for 96 h. Gene expression analysis showed an increase in the activity of stearoyl-CoA desaturase (Scd1) in the HC diet up to 69-fold (P < 0.0008) compared with the HF diet. This result was supported by the quantitative flux measurement of the isotopic incorporation of 2H into the respective cholesterol and cholesterol ester (CE) pools. We concluded that it is possible to readily obtain static and dynamic measurement of cholesterol and CEs in vivo by coupling novel LC/MS methods with stable isotope-based protocols. The liver plays a vital role in cholesterol metabolism (1Howles P.N. Cholesterol absorption and metabolism.Methods Mol. Biol. 2010; 602: 157-179Crossref PubMed Scopus (3) Google Scholar, 2Lecker J.L. Matthan N.R. Billheimer J.T. Rader D.J. Lichtenstein A.H. Impact of dietary fat type within the context of altered cholesterol homeostasis on cholesterol and lipoprotein metabolism in the F1B hamster.Metabolism. 2010; 59: 1491-1501Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 3Racette S.B. Lin X. Lefevre M. Spearie C.A. Most M.M. Ma L. Ostlund Jr, R.E. Dose effects of dietary phytosterols on cholesterol metabolism: a controlled feeding study.Am. J. Clin. Nutr. 2010; 91: 32-38Crossref PubMed Scopus (133) Google Scholar, 4Zhong S. Magnolo A.L. Sundaram M. Zhou H. Yao E.F. Di Leo E. Loria P. Wang S. Bamji-Mirza M. Wang L. Nonsynonymous mutations within APOB in human familial hypobetalipoproteinemia: evidence for feedback inhibition of lipogenesis and postendoplasmic reticulum degradation of apolipoprotein B.J. Biol. Chem. 2010; 285: 6453-6464Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 5Zhao C. Dahlman-Wright K. Liver X receptor in cholesterol metabolism.J. Endocrinol. 2010; 204: 233-240Crossref PubMed Scopus (326) Google Scholar) and homeostasis. In addition to this, production of bile acids from cholesterol also plays an important role in the secretion and degradation of plasma lipoproteins. A high level of cholesterol in the body circulation is strongly associated with atherosclerosis (6Alwaili K. Awan Z. Alshahrani A. Genest J. High-density lipoproteins and cardiovascular disease: 2010 update.Expert Rev. Cardiovasc. Ther. 2010; 8: 413-423Crossref PubMed Scopus (35) Google Scholar, 7Caserta C.A. Pendino G.M. Amante A. Vacalebre C. Fiorillo M.T. Surace P. Messineo A. Surace M. Alicante S. Cotichini R. Cardiovascular risk factors, nonalcoholic Fatty liver disease, and carotid artery intima-media thickness in an adolescent population in southern Italy.Am. J. Epidemiol. 2010; 171: 1195-1202Crossref PubMed Scopus (72) Google Scholar, 8Chakraborty S. Cai Y. Tarr M.A. Mapping oxidations of apo B-100 in human low density lipoprotein by LC-MS/MS.Anal. Biochem. 2010; : 109-117Crossref PubMed Scopus (14) Google Scholar, 9Gooding H.C. de Ferranti S.D. Cardiovascular risk assessment and cholesterol management in adolescents: getting to the heart of the matter.Curr. Opin. Pediatr. 2010; : 398-404Crossref PubMed Scopus (27) Google Scholar, 10Unverdorben M. von Holt K. Winkelmann B.R. Smoking and atherosclerotic cardiovascular disease: part II: role of cigarette smoking in cardiovascular disease development.Biomark Med. 2009; 3: 617-653Crossref PubMed Scopus (33) Google Scholar). The source of cholesterol comes from different areas: dietary, de novo synthesis, and synthesis in extrahepatic tissues. The liver acts as a cross-junction at which cholesterol is incorporated into HDL/LDL, and then secreted as free cholesterol in the bile or in the form of bile salts/acids. Studies of cholesterol metabolism typically require measurements of static concentrations of cholesterol to identify differences between models or to determine the presence or absence of a disease phenotype. The simultaneous use of stable or radio isotope flux analysis can aid in understanding the nature of a metabolic abnormality and yield information regarding the dynamics that contribute to altered or perturbed homeostasis. Questions surrounding cholesterol dynamics have been addressed using isotopic-labeled water for nearly 70 years, beginning with the pioneering studies of Schoenheimer (11Schoenheimer R. Rittenberg D. Deuterium as an indicator in the study of intermediary metabolism.Science. 1935; 82: 156-157Crossref PubMed Scopus (81) Google Scholar, 12Schoenheimer R. The investigation of intermediary metabolism with the aid of heavy hydrogen. Harvey Lecture, January 21, 1937.Bull. N. Y. Acad. Med. 1937; 13: 272-295PubMed Google Scholar, 13Schoenheimer R. Rittenberg D. The application of isotopes to the study of intermediary metabolism.Science. 1938; 87: 221-226Crossref PubMed Scopus (64) Google Scholar) based on the use of 2H2O and the classical work of Dietschy (14Dietschy J.M. Spady D.K. Measurement of rates of cholesterol synthesis using tritiated water.J. Lipid Res. 1984; 25: 1469-1476Abstract Full Text PDF PubMed Google Scholar, 15Dietschy J.M. Regulation of cholesterol metabolism in man and in other species.Klin. Wochenschr. 1984; 62: 338-345Crossref PubMed Scopus (85) Google Scholar, 16Dietschy J.M. Spady D.K. Regulation of low density lipoprotein uptake and degradation in different animals species.Agents Actions Suppl. 1984; 16: 177-190PubMed Google Scholar, 17Dietschy J.M. LDL cholesterol: its regulation and manipulation.Hosp. Pract. (Off. Ed.). 1990; 25: 67-78Crossref PubMed Scopus (4) Google Scholar, 18Dietschy J.M. Turley S.D. Spady D.K. Role of liver in the maintenance of cholesterol and low density lipoprotein homeostasis in different animal species, including humans.J. Lipid Res. 1993; 34: 1637-1659Abstract Full Text PDF PubMed Google Scholar, 19Dietschy J.M. Woollett L.A. Spady D.K. The interaction of dietary cholesterol and specific fatty acids in the regulation of LDL receptor activity and plasma LDL-cholesterol concentrations.Ann. N. Y. Acad. Sci. 1993; 676: 11-26Crossref PubMed Scopus (42) Google Scholar, 20Dietschy J.M. Theoretical considerations of what regulates low-density-lipoprotein and high-density-lipoprotein cholesterol.Am. J. Clin. Nutr. 1997; 65: 1581S-1589SCrossref PubMed Scopus (64) Google Scholar) et al. based on 3H2O. Considering the dose of radiation (typically in mCi) and the advances in mass spectrometry and related instrumentation (e.g., coupling to GC), it is not surprising that many investigators have turned their attention toward the use of the stable isotope 2H2O. The elegant work on human sterol and fatty acid (FA) flux carried out by Schoeller et al. (21Schoeller D.A. Uses of stable isotopes in the assessment of nutrient status and metabolism.Food Nutr. Bull. 2002; 23: 17-20Crossref PubMed Google Scholar), Wong et al. (22Wong W.W. Hachey D.L. Clarke L.L. Zhang S. Llaurador M. Pond W.G. An improved HPLC method to purify erythrocyte cholesterol for estimation of in vivo cholesterol synthesis using the deuterium method.Appl. Radiat. Isot. 1994; 45: 529-533Crossref PubMed Scopus (21) Google Scholar, 23Wong W.W. Hachey D.L. Feste A. Leggitt J. Clarke L.L. Pond W.G. Klein P.D. Measurement of in vivo cholesterol synthesis from 2H2O: a rapid procedure for the isolation, combustion, and isotopic assay of erythrocyte cholesterol.J. Lipid Res. 1991; 32: 1049-1056Abstract Full Text PDF PubMed Google Scholar), and Jones et al. (24Jones P.J. Schoeller D.A. Evidence for diurnal periodicity in human cholesterol synthesis.J. Lipid Res. 1990; 31: 667-673Abstract Full Text PDF PubMed Google Scholar, 25Jones P.J. Scanu A.M. Schoeller D.A. Plasma cholesterol synthesis using deuterated water in humans: effect of short-term food restriction.J. Lab. Clin. Med. 1988; 111: 627-633PubMed Google Scholar) has opened up the number of applications for the use of deuterated water in flux lipid experiments. Although the ability to couple static and dynamic measurements in studies of cholesterol metabolism is of obvious importance, most studies have been done under relatively low-level analytical resolution. For example, investigators typically examine the metabolism of free cholesterol and/or total cholesterol esters (CE). In addition, the classical GC/MS and GC-isotope ratio mass spectrometry (IRMS) methods are not suitable for routine use in high-throughput analyses because cholesterol esters are generally separated offline as a single pool (e.g., using TLC), and then subjected to saponification, extraction, derivatization, and finally mass spectrometry analysis. This is a more time consuming and laborious process, where given today's demands, especially in the discovery arena, it is essential to make decisions more quickly than ever. Recent advances in high-resolution LC-MS/MS have enabled the rapid high-throughput analyses of complex mixtures, which can be used to obtain information regarding different lipid classes and subclasses. These instruments are able to analyze complex biological mixtures with minimal sample preparation. A variety of different ionization techniques were utilized for the experiments described here. For example, cholesterol is particularly difficult to ionize by electrospray (ESI) mass spectrometry as its proton affinity is relatively low; however, ESI is suitable for measuring the ammoniated adduct of CE with good sensitivity. This finding is highlighted by a comparison of equimolar concentrations (1 µg/ml) of CE 16:0 and CE 18:0 analyzed by ESI and atmospheric pressure chemical ionization (APCI) (supplementary Fig. I). For both saturated CEs, the ESI technique proved to have a better signal intensity than APCI mode (∼7- to 13-fold better). Having said that, other researchers, including Butovich et al. (26Butovich I.A. Cholesteryl esters as a depot for very long chain fatty acids in human meibum.J. Lipid Res. 2009; 50: 501-513Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 27Butovich I.A. Uchiyama E. McCulley J.P. Lipids of human meibum: mass-spectrometric analysis and structural elucidation.J. Lipid Res. 2007; 48: 2220-2235Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar), have shown the application of APCI to measure in one analytical run free cholesterol and cholesterol esters coupled to a reversed phase column. But in their research, they were not measuring metabolic flux of free cholesterol and cholesterol esters in plasma. In our study, obtaining the best possible sensitivity was key to measuring the M1/M0 isotopomer ratio for free cholesterol and cholesterol esters. One ionization mode, such as APCI, was not sensitive enough to measure both analytes (free cholesterol and cholesterol esters) in one analytical run. In addition to the measurement of CEs by ESI, we investigated the use of atmospheric pressure photoionization (APPI) for the measurement of free cholesterol. The major advantage of APPI over APCI is that, for free cholesterol, we have experienced better limits of detection (between 3- and 4-fold better signal) than APCI mode (supplementary Fig. II). This technique has been used in the past to ionize less polar biochemicals, such as sterols and steroids (28Zhang F. Bartels M.J. Geter D.R. Carr M.S. McClymount L.E. Marino T.A. Klecka G.M. Simultaneous quantitation of testosterone, estradiol, ethinyl estradiol, and 11-ketotestosterone in fathead minnow fish plasma by liquid chromatography/positive atmospheric pressure photoionization tandem mass spectrometry.Rapid Commun. Mass Spectrom. 2009; 23: 3637-3646Crossref PubMed Scopus (35) Google Scholar, 29Karuna R. von Eckardstein A. Rentsch K.M. Dopant assisted-atmospheric pressure photoionization (DA-APPI) liquid chromatography-mass spectrometry for the quantification of 27-hydroxycholesterol in plasma.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 261-268Crossref PubMed Scopus (32) Google Scholar, 30Cai S.S. Syage J.A. Hanold K.A. Balogh M.P. Ultra performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry for high-sensitivity and high-throughput analysis of U.S. Environmental Protection Agency 16 priority pollutants polynuclear aromatic hydrocarbons.Anal. Chem. 2009; 81: 2123-2128Crossref PubMed Scopus (122) Google Scholar, 31Cai Y. McConnell O. Bach 2nd, A.C. Suitability of tetrahydofuran as a dopant and the comparison to other existing dopants in dopant-assisted atmospheric pressure photoionization mass spectrometry in support of drug discovery.Rapid Commun. Mass Spectrom. 2009; 23: 2283-2291Crossref PubMed Scopus (13) Google Scholar, 32Borges N.C. Astigarraga R.B. Sverdloff C.E. Galvinas P.R. da Silva W.M. Rezende V.M. Moreno R.A. A novel and sensitive method for ethinylestradiol quantification in human plasma by high-performance liquid chromatography coupled to atmospheric pressure photoionization (APPI) tandem mass spectrometry: application to a comparative pharmacokinetics study.J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 2009; 877: 3601-3609Crossref PubMed Scopus (15) Google Scholar, 33Robb D.B. Blades M.W. State-of-the-art in atmospheric pressure photoionization for LC/MS.Anal. Chim. Acta. 2008; 627: 34-49Crossref PubMed Scopus (121) Google Scholar, 34McCulloch R.D. Robb D.B. Blades M.W. A dopant introduction device for atmospheric pressure photoionization with liquid chromatography/mass spectrometry.Rapid Commun. Mass Spectrom. 2008; 22: 3549-3554Crossref PubMed Scopus (5) Google Scholar). The ionization mechanism is somewhat different from electrospray because in most cases a dopant is used to provide the proton to complete the ionization process. Typical dopants that may be used are acetone and toluene. The ionization is initiated by 10-eV photons emitted by a krypton discharge lamp. The mechanism of ionization by APPI involves the absorption of photons by the molecule(s) to be analyzed, followed by the ejection of an electron resulting in a molecular cation M+. This reaction only occurs if the ionization energy of the dopant is lower than the ionization energy of the photons. The dopant provides the proton, and the radical cation will react with the dopant to form a stable [M+H]+ cation. We report here on a simple method(s) for dissecting cholesterol metabolism via LC-MS/MS. We aimed to determine if we could simultaneously quantify the abundance and the isotopic labeling following 2H2O administration (35Previs S.F. Hazey J.W. Diraison F. Beylot M. David F. Brunengraber H. Assay of the deuterium enrichment of water via acetylene.J. Mass Spectrom. 1996; 31: 639-642Crossref PubMed Scopus (57) Google Scholar, 36Previs S.F. Fatica R. Chandramouli V. Alexander J.C. Brunengraber H. Landau B.R. Quantifying rates of protein synthesis in humans by use of 2H2O: application to patients with end-stage renal disease.Am. J. Physiol. Endocrinol. Metab. 2004; 286: E665-E672Crossref PubMed Scopus (83) Google Scholar) of different CE species in the presence of a high-carbohydrate (HC) or high-fat (HF) diet. Male C57Bl/6 mice from Taconic were acclimated in the animal facility for one week. At an age of 10-weeks-old, mice were randomized into two groups (n = 26 per group), and the diet was switched to either a high carbohydrate (HC) diet (D12450, 10% fat, 70% carbohydrate, and 20% protein, Research Diets, NJ) or a carbohydrate-free (CF) diet (D12369B, 90% fat, 0% carbohydrate, and 10% protein, Research Diets, NJ). The diet intervention proceeded for 13 days, and then all mice were then given an intraperitoneal injection of labeled water (20 ml/kg of body weight, 99% 2H2O). After injection, mice were returned to their cages (n = 6 mice per cage) and maintained on 5% 2H-labeled drinking water for the remainder of the study. This design is sufficient to maintain a steady-state 2H labeling of body water. Mice in each group were fed the respective diets ad libitum. They were sedated on various days after injection (n = 6 per day per group), blood and tissue samples (liver tissue was used for gene expression analysis only) were then collected and quick-frozen in liquid nitrogen. Samples were stored at −80°C until analyzed. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Merck Research Laboratories (Rahway, NJ). The 2H-labeling of plasma water was determined as described by Shah et al. (37Shah V. Herath K. Previs S.F. Hubbard B.K. Roddy T.P. Headspace analyses of acetone: a rapid method for measuring the (2)H-labeling of water.Anal Biochem. 2010; 404: 235-237Crossref PubMed Scopus (49) Google Scholar). Briefly, 2H present in water is exchanged with hydrogen bound to acetone by incubating samples (e.g., 10 μl of plasma or known standards) in a 2 ml glass screw-top GC vial at room temperature for 4 h with 2 μl 10N NaOH and 5 μl of acetone. The instrument is programmed to inject 5 μl of headspace gas from the GC vial in a splitless mode. Samples were analyzed using a 0.8 min isothermal run (Agilent 5973 MS coupled to a 6890 GC oven-fitted with a DB-17 MS column, 15 m × 250 µm × 0.15 µm; the oven was set at 170°C; and helium carrier flow was set at 1.0 ml × min−1), acetone eluted at ∼0.4 min, and the mass spectrometer was set to perform selected ion monitoring of m/z 58 and 59 (10 ms dwell time per ion) in the electron impact ionization mode. Plasma samples for GC/MS analysis were processed in 1.5 ml eppendorff tubes. An amount of 25 μl internal standard (FA 17:0, 0.5 mg/ml CHCl3) and 100 μl 1N KOH in 80% ethanol was added to 50 μl of plasma. The samples were heated at 65°C for 1 h. Samples were acidified with 25 μl 6N HCl and extracted in 125 μl chloroform followed by vigorous vortexing for 20 s The samples were centrifuged at 3000 rpm for 5 min, and then 100 μl of chloroform (lower layer) was collected and evaporated to dryness under N2. Samples were derivatized using bis trimethyl silyl trifluoroacetamide (BSTFA) plus 10% trimethylchlorosilane (TMCS), 50 μl was added to the sample, and then it was incubated at 75°C for 1 h. Excess BSTFA was evaporated to dryness in N2. The TMS derivative was reconstituted in 50 μl ethyl acetate for analysis by GC/MS. Samples were analyzed by GC/MS using the Agilent 6890 gas chromatograph linked to an Agilent 5973 mass selective detector (MSD) (Agilent, Palo Alto, CA) operated at 70 eV. Gas chromatography was performed using an Agilent J and W DB-5MS capillary column (30.0 m × 250 μm × 0.25 μm). An amount of 2 μl was injected in a 20:1 split. The inlet temperature was set at 250°C and the helium gas carrier flow was set at 1 ml/min−1. The oven temperature was started at 150°C, raised at 20°C per min to 310°C, and held at this temperature for 6 min. The MSD was set for selected ion monitoring (SIM) of m/z 313, 314 for the palmitate TMS derivative; 327, 328, 329 for heptadecanoic acid TMS derivative; and 368, 369 for cholesterol TMS derivative with 10 ms dwell time per ion. Concentrations of fatty acids/cholesterol were corrected for by a standard curve with varying combinations of fatty acid or cholesterol with their respective D1-derivatives. Plasma samples from each animal (20 μl) were extracted for lipid analysis by LC/MS-MS using a dichloromethane (DCM)/methanol mixture (2:1, v/v) in accordance with the method described by Bligh and Dyer (38Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (43132) Google Scholar, 39Castro-Perez J.M. Kamphorst J. DeGroot J. Lafeber F. Goshawk J. Yu K. Shockcor J.P. Vreeken R.J. Hankemeier T. Comprehensive LC-MS E lipidomic analysis using a shotgun approach and its application to biomarker detection and identification in osteoarthritis patients.J. Proteome Res. 2010; 9: 2377-2389Crossref PubMed Scopus (184) Google Scholar). During the procedure, the samples were spiked with non-naturally occurring and deuterated lipid internal standards (17:0 containing CE and D6-cholesterol; Sigma Aldrich, St Louis, MO) in final concentrations of 2 µg/ml. The inlet system was composed of an Acquity UPLC (Waters, Milford, MA). Mouse plasma lipid extracts were injected (10 µL) onto a 1.8 µm particle 100 × 2.1 mm id Waters Acquity HSS T3 column (Waters); the column was maintained at 55°C. The flow rate used for these experiments was 0.4 ml/min. A binary gradient system consisting of acetonitrile (Burdick and Jackson, USA) and water with 10 mM ammonium formate (Sigma-Aldrich) (40:60, v/v) was used as eluent A. Eluent B consisted of acetonitrile and isopropanol (Burdick and Jackson) containing 10 mM ammonium formate (10:90, v/v). The sample analysis was performed by using a linear gradient (curve 6) over a 15 min total run time. During the initial portion of the gradient, it was held at 60% A and 40% B. For the next 10 min, the gradient was ramped in a linear fashion to 100% B and held at this composition for 2 min. Then the system was switched back to 60% B and 40% A and equilibrated for an additional 3 min. For the free cholesterol measurements by LC/APPI-MS, the gradient conditions were identical apart from the fact that no ammonium formate was used as the additive. The inlet system was directly coupled to a hybrid quadrupole orthogonal time-of-flight mass spectrometer (SYNAPT G2 HDMS, Waters, MS Technologies, Manchester, UK). Electrospray positive and APPI positive ionization modes were used. In ESI mode, a capillary voltage and cone voltage of ±2 kV and ±30 V, respectively, was used. The desolvation source conditions were as follows: for the desolvation gas, 700 l/hr was used and the desolvation temperature was kept at 450C. APPI was utilized using a krypton discharge lamp (10-eV photons) set with a repeller voltage of ±3.5 kV. The dopant used for the APPI experiments was acetone (Fisher Scientific, Pittsburgh, PA), which was infused at a continuous flow rate of 100 μl/min post column. The desolvation source conditions for APPI were as follows: for the desolvation gas, 900 l/hr was used and the desolvation temperature was kept at 600C. Data were acquired over the mass range of 50-1200 Da for both MS and MSE modes (40Wrona M. Mauriala T. Bateman K.P. Mortishire-Smith R.J. O'Connor D. ‘All-in-one’ analysis for metabolite identification using liquid chromatography/hybrid quadrupole time-of-flight mass spectrometry with collision energy switching.Rapid Commun. Mass Spectrom. 2005; 19: 2597-2602Crossref PubMed Scopus (155) Google Scholar, 41Bateman K.P. Castro-Perez J. Wrona M. Shockcor J.P. Yu K. Oballa R. Nicoll-Griffith D.A. MSE with mass defect filtering for in vitro and in vivo metabolite identification.Rapid Commun. Mass Spectrom. 2007; 21: 1485-1496Crossref PubMed Scopus (222) Google Scholar, 42Castro-Perez J.M. Current and future trends in the application of HPLC-MS to metabolite-identification studies.Drug Discov. Today. 2007; 12: 249-256Crossref PubMed Scopus (90) Google Scholar, 43Plumb R.S. Johnson K.A. Rainville P. Smith B.W. Wilson I.D. Castro-Perez J.M. Nicholson J.K. UPLC/MS(E), a new approach for generating molecular fragment information for biomarker structure elucidation.Rapid Commun. Mass Spectrom. 2006; 20: 1989-1994Crossref PubMed Scopus (393) Google Scholar). The mass spectral resolution was set to 25K full width half mass (FWHM) and typical mass accuracies were in the order of 0-2 ppm. The system was equipped with an integral LockSpray unit with its own reference sprayer that was controlled automatically by the acquisition software to collect a reference scan every 10 s lasting 0.3 s. The LockSpray internal reference used for these experiments was Leucine enkephalin (Sigma-Aldrich) at a concentration of 5 ng/μl in 50% acetonitrile/50% H2O plus 0.1% formic acid (v/v). The reference internal calibrant was introduced into the lock mass sprayer at a constant flow rate of 50 µl/min using an integrated solvent delivery pump. A single lock mass calibration at m/z 556.2771 in positive ion mode was used during analysis. The mass spectrometer was operated in the MSE mode of acquisition. During this acquisition method, the first quadrupole Q1 is operated in a wide band RF mode only, allowing all ions to enter the T-wave collision cell. Two discrete and independent interleaved acquisitions functions are automatically created. The first function, typically set at 5 eV, collects low energy or unfragmented data, and the second function collects high energy or fragmented data typically set by using a collision energy ramp from 25-35 eV. In both instances, argon gas is used for collision-induced dissociation (CID). This mode of operation allows for fragmentation ions to be generated ad hoc, and the use of the software data mining tool allowed for the alignment of the low and high energy data. This mode of acquisition proved to be adequate for high throughput screening, but there are some cases where complete ion coelution occurs between the low and high energy acquisitions. When this occurred, more rigorous fragmentation MS/MS CID experiments were utilized. For the LC/MS and GC/MS analysis of the isotopic dilution of cholesterol, a set of standards were prepared in chloroform (1 mg/ml), ranging from 0% excess 2H-labeling up to 2.5% excess 2H-labeling (0%, 0.15%, 0.3%, 0.6%, 1.25%, 2.5%) by mixing with cholesterol and its D1-derivative. Labeled palmitate was prepared in the same fashion as cholesterol for the GC/MS analysis only, and heptadecanoic acid was used as the internal standard. All the samples were diluted 10-fold with (65:5:30 v/v/v) IPA:MeOH:H2O to achieve a final concentration of 0.1 mg/ml. For GC/MS analysis 10 μl of each standard was derivatized with BSTFA as described above. For the quantitation of the contribution of cholesterol synthesis, the data was processed using a precursor:product labeling ratio to the general equation: % newly made material = product labeling / (water labeling × n) × 100, where n is the number of exchangeable hydrogen, assumed to equal 26, and the product labeling is determined from the ratio of M1/M0 isotopomers (44Diraison F. Pachiaudi C. Beylot M. In vivo measurement of plasma cholesterol and fatty acid synthesis with deuterated water: determination of the average number of deuterium atoms incorporated.Metabolism. 1996; 45: 817-821Abstract Full Text PDF PubMed Scopus (77) Google Scholar). The GC/MS and LC/MS data acquired were processed by the instrument manufacturer’s software (ChemStation and MassLynx, respectively). Gene expression data were processed using Ingenuity software (Ingenuity Systems, Redwood City, CA). For statistical analysis, all data are presented as ± SEM. Differences between groups were computed by Student’s t-test (GraphPad Prism, La Jolla, CA). Post-test analysis for quantifiable variables was conducted using Mann-Whitney U nonparametric test with two-tailed P values. P < 0.05 was considered statistically significant for all data derived from the experiments. Liver tissue (∼20 mg) was snap-frozen in liquid nitrogen and stored at −80°C. The tissues were homogenized in 600 μl RLT lysis buffer (Qiagen, Valencia, CA) containing 0.1% (v/v) β-mercaptoethanol using a PowerGen 125 homogenizer and 7 × 65 mm disposable plastic generators (Fisher Scientific). Total RNA was extracted from the homogenized tissue using RNeasy Mini Kit (Qiagen, Valencia, CA) following the manufacturer's protocol. cDNA was generated from 2 μg of RNA using RT2 First Strand kit (SA Biosciences). Real-time PCR analysis was performed on the 7900HT PCR System (Applied Biosystems, Foster City, CA) with 2× SYBR PCR Master Mix and mouse-specific PCR primers for mouse Scd1, (SABiosciences). Expression levels of stearoyl-CoA desaturase (Scd1) mRNA were normalized to an average of that of mouse β-actin (Actb), glyceraldehyde 3-phosphate dehydrogenase (Gapdh), beta-glucuronidase (Gusb), hypoxanthine-guanine phosphoribosyltransferase (Hprt1), peptidylprolyl isomerase A (cyclophilin A) (Ppia), and ribosomal protein 113a (Rp113a) in each sample. The water labeling after 3 h was kept constant for both diets for the duration of the study at ∼2.5% (Fig. 1). Gene expression results from the HC diet revealed an upregulation in the expression of sterol-regulatory element binding protein (SREBP)1c" @default.
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• W2151025672 date "2011-01-01" @default.
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• W2151025672 title "In vivo D2O labeling to quantify static and dynamic changes in cholesterol and cholesterol esters by high resolution LC/MS" @default.
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