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William J Griffiths - One of the best experts on this subject based on the ideXlab platform.
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Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry
MDPI AG, 2019Co-Authors: Jonas Abdel-khalik, Yuqin Wang, Peter J. Crick, Eylan Yutuc, William J GriffithsAbstract:Enzyme-assisted derivatization for Sterol Analysis (EADSA) is a technology designed to enhance sensitivity and specificity for Sterol Analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on Sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial choleSterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for choleSterol oxidase, making the method applicable to Sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the Sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]+) upon electrospray ionization and informative fragmentation spectra. The method shows promise for Analysis of bile alcohols and 3α-hydroxy-5β-C27-acids, enhancing the range of Sterols that can be analyzed at high sensitivity in Sterolomic studies
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revised sample preparation for the Analysis of oxySterols by enzyme assisted derivatisation for Sterol Analysis eadsa
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Peter J. Crick, Yuqin Wang, William T Bentley, William J GriffithsAbstract:Sterols, and specifically oxySterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxySterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography–mass spectrometry method termed enzyme-assisted derivatisation for Sterol Analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate choleSterol from oxySterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic–lipophilic balanced reversed-phase cartridges.
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analytical strategies for characterization of oxySterol lipidomes liver x receptor ligands in plasma
Free Radical Biology and Medicine, 2013Co-Authors: William J Griffiths, Peter J. Crick, Yuchen Wang, Michael Ogundare, Karin Tuschl, Andrew A M Morris, Brian W Bigger, Peter E Clayton, Yuqin WangAbstract:Bile acids, bile alcohols, and hormonal steroids represent the ultimate biologically active products of choleSterol metabolism in vertebrates. However, intermediates in their formation, including oxySterols and cholestenoic acids, also possess known, e.g., as ligands to nuclear and G-protein-coupled receptors, and unknown regulatory activities. The potential diversity of molecules originating from the choleSterol structure is very broad and their abundance in biological materials ranges over several orders of magnitude. Here we describe the application of enzyme-assisted derivatization for Sterol Analysis (EADSA) in combination with liquid chromatography-electrospray ionization-mass spectrometry to define the oxySterol and cholestenoic acid metabolomes of human plasma. Quantitative profiling of adult plasma using EADSA leads to the detection of over 30 metabolites derived from choleSterol, some of which are ligands to the nuclear receptors LXR, FXR, and pregnane X receptor or the G-protein-coupled receptor Epstein-Barr virus-induced gene 2. The potential of the EADSA technique in screening for inborn errors of choleSterol metabolism and biosynthesis is demonstrated by the unique plasma profile of patients suffering from cerebrotendinous xanthomatosis. The analytical methods described are easily adapted to the Analysis of other biological fluids, including cerebrospinal fluid, and also tissues, e.g., brain, in which nuclear and G-protein-coupled receptors may have important regulatory roles.
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potential of Sterol Analysis by liquid chromatography tandem mass spectrometry for the prenatal diagnosis of smith lemli opitz syndrome
Clinical Chemistry, 2008Co-Authors: William J Griffiths, Yuqin Wang, Kersti Karu, Emmanuel Samuel, Shane Mcdonnell, Martin HornshawAbstract:Background: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of choleSterol synthesis, is classically diagnosed prenatally by GC-MS Analysis of Sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder. Methods: 3β-HydroxySterols in amniotic fluid are oxidized with choleSterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a “one-pot” reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)–MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots. Results: In a double-blind Analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholeSterol (7 + 8-DHC) to choleSterol was 0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column. Conclusions: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
Henning Hamm - One of the best experts on this subject based on the ideXlab platform.
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conradi hunermann happle syndrome x linked dominant chondrodysplasia punctata confirmed by plasma Sterol and mutation Analysis
Acta Dermato-venereologica, 2008Co-Authors: Annette Kolbmaurer, Karlheinz Grzeschik, Dorothea Haas, Evabettina Brocker, Henning HammAbstract:Conradi-Hunermann-Happle syndrome, or X-linked dominant chondrodysplasia punctata, is a rare genetic disorder characterized by skeletal dysplasia, stippled epiphyses, cataracts, transient ichthyosis and atrophic residua in a mosaic pattern. Mutations in the gene encoding the emopamil-binding protein have been identified as an underlying cause. A 5-year-old girl presented for evaluation of ill-defined patches of cicatricial alopecia. In addition, subtle follicular atrophoderma, esotropia, craniofacial asymmetry and short stature were noted. Her history revealed widespread scaly erythema and eye surgery for congenital cataract in the first months of life. Diagnosis of Conradi-Hunermann-Happle syndrome was confirmed by plasma Sterol Analysis showing markedly elevated levels of 8(9)-cholestenol and 8-dehydrocholeSterol and by detection of a missense mutation (c.307G>A; p.E103K) in the emopamil-binding protein gene. We suggest that plasma Sterol Analysis is a reliable method of establishing the diagnosis of Conradi-Hunermann-Happle syndrome, even in patients with less striking phenotypical changes beyond infancy.
Yuqin Wang - One of the best experts on this subject based on the ideXlab platform.
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Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry
MDPI AG, 2019Co-Authors: Jonas Abdel-khalik, Yuqin Wang, Peter J. Crick, Eylan Yutuc, William J GriffithsAbstract:Enzyme-assisted derivatization for Sterol Analysis (EADSA) is a technology designed to enhance sensitivity and specificity for Sterol Analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on Sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial choleSterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for choleSterol oxidase, making the method applicable to Sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the Sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]+) upon electrospray ionization and informative fragmentation spectra. The method shows promise for Analysis of bile alcohols and 3α-hydroxy-5β-C27-acids, enhancing the range of Sterols that can be analyzed at high sensitivity in Sterolomic studies
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revised sample preparation for the Analysis of oxySterols by enzyme assisted derivatisation for Sterol Analysis eadsa
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Peter J. Crick, Yuqin Wang, William T Bentley, William J GriffithsAbstract:Sterols, and specifically oxySterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxySterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography–mass spectrometry method termed enzyme-assisted derivatisation for Sterol Analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate choleSterol from oxySterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic–lipophilic balanced reversed-phase cartridges.
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analytical strategies for characterization of oxySterol lipidomes liver x receptor ligands in plasma
Free Radical Biology and Medicine, 2013Co-Authors: William J Griffiths, Peter J. Crick, Yuchen Wang, Michael Ogundare, Karin Tuschl, Andrew A M Morris, Brian W Bigger, Peter E Clayton, Yuqin WangAbstract:Bile acids, bile alcohols, and hormonal steroids represent the ultimate biologically active products of choleSterol metabolism in vertebrates. However, intermediates in their formation, including oxySterols and cholestenoic acids, also possess known, e.g., as ligands to nuclear and G-protein-coupled receptors, and unknown regulatory activities. The potential diversity of molecules originating from the choleSterol structure is very broad and their abundance in biological materials ranges over several orders of magnitude. Here we describe the application of enzyme-assisted derivatization for Sterol Analysis (EADSA) in combination with liquid chromatography-electrospray ionization-mass spectrometry to define the oxySterol and cholestenoic acid metabolomes of human plasma. Quantitative profiling of adult plasma using EADSA leads to the detection of over 30 metabolites derived from choleSterol, some of which are ligands to the nuclear receptors LXR, FXR, and pregnane X receptor or the G-protein-coupled receptor Epstein-Barr virus-induced gene 2. The potential of the EADSA technique in screening for inborn errors of choleSterol metabolism and biosynthesis is demonstrated by the unique plasma profile of patients suffering from cerebrotendinous xanthomatosis. The analytical methods described are easily adapted to the Analysis of other biological fluids, including cerebrospinal fluid, and also tissues, e.g., brain, in which nuclear and G-protein-coupled receptors may have important regulatory roles.
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potential of Sterol Analysis by liquid chromatography tandem mass spectrometry for the prenatal diagnosis of smith lemli opitz syndrome
Clinical Chemistry, 2008Co-Authors: William J Griffiths, Yuqin Wang, Kersti Karu, Emmanuel Samuel, Shane Mcdonnell, Martin HornshawAbstract:Background: Smith-Lemli-Opitz syndrome (SLOS), a severe disorder of choleSterol synthesis, is classically diagnosed prenatally by GC-MS Analysis of Sterols in amniotic fluid. Considering the current trend toward tandem mass spectrometry (MS/MS) methodologies, we developed prototype LC-MS/MS methods for accurate diagnosis of the disorder. Methods: 3β-HydroxySterols in amniotic fluid are oxidized with choleSterol oxidase to their corresponding 3-ketones, which are then derivatized with Girard P (GP) hydrazine in a “one-pot” reaction. The resulting GP-hydrazones give an improved response in electrospray (ES)–MS/MS owing to the presence of a charged quaternary nitrogen and are analyzed by reversed-phase LC-ES-MS/MS. Both capillary and conventional LC-MS/MS formats are suitable, and the method is also applicable to paper-absorbed blood spots. Results: In a double-blind Analysis of 18 amniotic fluid samples comprising 6 SLOS and 12 controls, the ratio of 7 + 8-dehydrocholeSterol (7 + 8-DHC) to choleSterol was 0.20 [0.20–1.13, 0.79 (0.35)] in SLOS (intraassay variation 4.56%), corresponding to a difference in ratios between the 2 groups of at least a factor of 10. The limit of quantification was equivalent to that of 2 nL amniotic fluid injected on-column. Conclusions: We describe a proof-of-concept for the prenatal diagnosis of SLOS. Further developments will be necessary to automate sample handling and reduce chromatographic time for the methodology to be used in pre- and postnatal diagnosis.
Zhenyu Chen - One of the best experts on this subject based on the ideXlab platform.
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plasma choleSterol lowering activity of gingerol and shogaol enriched extract is mediated by increasing Sterol excretion
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Xiaobo Wang, Yuk Man Li, Rui Jiao, Ka Ying, Shengmin Sang, Yu Huang, Lijun Wang, Zhenyu ChenAbstract:The present study investigated the choleSterol-lowering activity of gingerol- and shogaol-enriched ginger extract (GSE). Thirty hamsters were divided into three groups and fed the control diet or one of the two experimental diets containing 0.5 and 1.0% GSE. Plasma total choleSterol, liver choleSterol, and aorta atherosclerotic plaque were dose-dependently decreased with increasing amounts of GSE added into diets. The fecal Sterol Analysis showed dietary GSE increased the excretion of both neutral and acidic Sterols in a dose-dependent manner. GSE down-regulated the mRNA levels of intestinal Niemann–Pick C1-like 1 protein (NPC1L1), acyl CoA:choleSterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP binding cassette transporter 5 (ABCG5), whereas it up-regulated hepatic choleSterol-7α-hydroxylase (CYP7A1). It was concluded that beneficial modification of the lipoprotein profile by dietary GSE was mediated by enhancing excretion of fecal choleSterol and bile acids via...
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plasma choleSterol lowering activity of gingerol and shogaol enriched extract is mediated by increasing Sterol excretion
Journal of Agricultural and Food Chemistry, 2014Co-Authors: Lin Lei, Rui Jiao, Ka Ying, Shengmin Sang, Yu Huang, Xiaobo Wang, Lijun Wang, Yuwei Liu, Sun Wa Man, Zhenyu ChenAbstract:The present study investigated the choleSterol-lowering activity of gingerol- and shogaol-enriched ginger extract (GSE). Thirty hamsters were divided into three groups and fed the control diet or one of the two experimental diets containing 0.5 and 1.0% GSE. Plasma total choleSterol, liver choleSterol, and aorta atherosclerotic plaque were dose-dependently decreased with increasing amounts of GSE added into diets. The fecal Sterol Analysis showed dietary GSE increased the excretion of both neutral and acidic Sterols in a dose-dependent manner. GSE down-regulated the mRNA levels of intestinal Niemann-Pick C1-like 1 protein (NPC1L1), acyl CoA:choleSterol acyltransferase 2 (ACAT2), microsomal triacylglycerol transport protein (MTP), and ATP binding cassette transporter 5 (ABCG5), whereas it up-regulated hepatic choleSterol-7α-hydroxylase (CYP7A1). It was concluded that beneficial modification of the lipoprotein profile by dietary GSE was mediated by enhancing excretion of fecal choleSterol and bile acids via up-regulation of hepatic CYP7A1 and down-regulation of mRNA of intestinal NPC1L1, ACAT2, and MTP.
Peter J. Crick - One of the best experts on this subject based on the ideXlab platform.
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Developing an Enzyme-Assisted Derivatization Method for Analysis of C27 Bile Alcohols and Acids by Electrospray Ionization-Mass Spectrometry
MDPI AG, 2019Co-Authors: Jonas Abdel-khalik, Yuqin Wang, Peter J. Crick, Eylan Yutuc, William J GriffithsAbstract:Enzyme-assisted derivatization for Sterol Analysis (EADSA) is a technology designed to enhance sensitivity and specificity for Sterol Analysis using electrospray ionization⁻mass spectrometry. To date it has only been exploited on Sterols with a 3β-hydroxy-5-ene or 3β-hydroxy-5α-hydrogen structure, using bacterial choleSterol oxidase enzyme to convert the 3β-hydroxy group to a 3-oxo group for subsequent derivatization with the positively charged Girard hydrazine reagents, or on substrates with a native oxo group. Here we describe an extension of the technology by substituting 3α-hydroxysteroid dehydrogenase (3α-HSD) for choleSterol oxidase, making the method applicable to Sterols with a 3α-hydroxy-5β-hydrogen structure. The 3α-HSD enzyme works efficiently on bile alcohols and bile acids with this stereochemistry. However, as found by others, derivatization of the resultant 3-oxo group with a hydrazine reagent does not go to completion in the absence of a conjugating double bond in the Sterol structure. Nevertheless, Girard P derivatives of bile alcohols and C27 acids give an intense molecular ion ([M]+) upon electrospray ionization and informative fragmentation spectra. The method shows promise for Analysis of bile alcohols and 3α-hydroxy-5β-C27-acids, enhancing the range of Sterols that can be analyzed at high sensitivity in Sterolomic studies
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revised sample preparation for the Analysis of oxySterols by enzyme assisted derivatisation for Sterol Analysis eadsa
Analytical and Bioanalytical Chemistry, 2015Co-Authors: Peter J. Crick, Yuqin Wang, William T Bentley, William J GriffithsAbstract:Sterols, and specifically oxySterols, play important roles in the biosynthesis of bile acids and steroid hormones as well as possessing biological activities in their own right. Analysis of oxySterols is complicated due to their low abundance in biological systems and poor ionisation characteristics in mass spectrometry. Over the past decade, we have developed a liquid chromatography–mass spectrometry method termed enzyme-assisted derivatisation for Sterol Analysis (EADSA). Our derivatisation procedure relies on two solid-phase extraction steps to (i) separate choleSterol from oxySterols and (ii) remove excess derivatisation reagents. Recent inter-batch variation in C18 reversed-phase cartridges has led us to experiment with alternative columns. Here, we present our findings and report an improved sample preparation procedure using polymeric hydrophilic–lipophilic balanced reversed-phase cartridges.
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quantitative charge tags for Sterol and oxySterol Analysis
Clinical Chemistry, 2015Co-Authors: Peter J. Crick, William T Bentley, Andrew A M Morris, Brian W Bigger, Peter E Clayton, Jonas Abdelkhalik, Ian Matthews, Chiara Zerbinati, Luigi Tritapepe, Luigi IulianoAbstract:Background: Global Sterol Analysis is challenging owing to the extreme diversity of Sterol natural products, the tendency of choleSterol to dominate in abundance over all other Sterols, and the structural lack of a strong chromophore or readily ionized functional group. We developed a method to overcome these challenges by using different isotope-labeled versions of the Girard P reagent (GP) as quantitative charge-tags for the LC-MS Analysis of Sterols including oxySterols. Methods: Sterols/oxySterols in plasma were extracted in ethanol containing deuterated internal standards, separated by C18 solid-phase extraction, and derivatized with GP, with or without prior oxidation of 3β-hydroxy to 3-oxo groups. Results: By use of different isotope-labeled GPs, it was possible to analyze in a single LC-MS Analysis both Sterols/oxySterols that naturally possess a 3-oxo group and those with a 3β-hydroxy group. Intra- and interassay CVs were <15%, and recoveries for representative oxySterols and cholestenoic acids were 85%–108%. By adopting a multiplex approach to isotope labeling, we analyzed up to 4 different samples in a single run. Using plasma samples, we could demonstrate the diagnosis of inborn errors of metabolism and also the export of oxySterols from brain via the jugular vein. Conclusions: This method allows the profiling of the widest range of Sterols/oxySterols in a single analytical run and can be used to identify inborn errors of choleSterol synthesis and metabolism.
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analytical strategies for characterization of oxySterol lipidomes liver x receptor ligands in plasma
Free Radical Biology and Medicine, 2013Co-Authors: William J Griffiths, Peter J. Crick, Yuchen Wang, Michael Ogundare, Karin Tuschl, Andrew A M Morris, Brian W Bigger, Peter E Clayton, Yuqin WangAbstract:Bile acids, bile alcohols, and hormonal steroids represent the ultimate biologically active products of choleSterol metabolism in vertebrates. However, intermediates in their formation, including oxySterols and cholestenoic acids, also possess known, e.g., as ligands to nuclear and G-protein-coupled receptors, and unknown regulatory activities. The potential diversity of molecules originating from the choleSterol structure is very broad and their abundance in biological materials ranges over several orders of magnitude. Here we describe the application of enzyme-assisted derivatization for Sterol Analysis (EADSA) in combination with liquid chromatography-electrospray ionization-mass spectrometry to define the oxySterol and cholestenoic acid metabolomes of human plasma. Quantitative profiling of adult plasma using EADSA leads to the detection of over 30 metabolites derived from choleSterol, some of which are ligands to the nuclear receptors LXR, FXR, and pregnane X receptor or the G-protein-coupled receptor Epstein-Barr virus-induced gene 2. The potential of the EADSA technique in screening for inborn errors of choleSterol metabolism and biosynthesis is demonstrated by the unique plasma profile of patients suffering from cerebrotendinous xanthomatosis. The analytical methods described are easily adapted to the Analysis of other biological fluids, including cerebrospinal fluid, and also tissues, e.g., brain, in which nuclear and G-protein-coupled receptors may have important regulatory roles.