Peter J Crick1, T William Bentley1, Jonas Abdel-Khalik1, Ian Matthews2, Peter T Clayton3, Andrew A Morris4, Brian W Bigger5, Chiara Zerbinati6, Luigi Tritapepe7, Luigi Iuliano6, Yuqin Wang8, William J Griffiths8. 1. College of Medicine and. 2. College of Engineering, Swansea University, Swansea, U.K.; 3. Centre for Translational Omics, University College London Institute of Child Health, London, U.K.; 4. Willink Biochemical Genetics Unit, Manchester Centre for Genomic Medicine, St Mary's Hospital, Manchester, U.K.; 5. Stem Cell & Neurotherapies, Manchester Centre for Genomic Medicine, University of Manchester, Manchester, U.K.; 6. Department of Medico-Surgical Sciences and Biotechnology, Sapienza University of Rome, Latina, Italy; 7. Department of Anesthesiology and Intensive Care, Sapienza University of Rome, Rome, Italy. 8. College of Medicine and w.j.griffiths@swansea.ac.uk y.wang@swansea.ac.uk.
Abstract
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.
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.
Authors: William J Griffiths; Jonas Abdel-Khalik; Peter J Crick; Michael Ogundare; Cedric H Shackleton; Karin Tuschl; Mei Kwun Kwok; Brian W Bigger; Andrew A Morris; Akira Honda; Libin Xu; Ned A Porter; Ingemar Björkhem; Peter T Clayton; Yuqin Wang Journal: J Steroid Biochem Mol Biol Date: 2016-03-11 Impact factor: 4.292
Authors: Luigi Iuliano; Peter J Crick; Chiara Zerbinati; Luigi Tritapepe; Jonas Abdel-Khalik; Marc Poirot; Yuqin Wang; William J Griffiths Journal: Steroids Date: 2015-02-07 Impact factor: 2.668
Authors: Jonas Abdel-Khalik; Eylan Yutuc; Peter J Crick; Jan-Åke Gustafsson; Margaret Warner; Gustavo Roman; Kevin Talbot; Elizabeth Gray; William J Griffiths; Martin R Turner; Yuqin Wang Journal: J Lipid Res Date: 2016-11-03 Impact factor: 5.922
Authors: Peter J Crick; William J Griffiths; Juan Zhang; Martin Beibel; Jonas Abdel-Khalik; Jens Kuhle; Andreas W Sailer; Yuqin Wang Journal: Mol Neurobiol Date: 2016-11-23 Impact factor: 5.590
Authors: Peter J Crick; Lien Beckers; Myriam Baes; Paul P Van Veldhoven; Yuqin Wang; William J Griffiths Journal: Steroids Date: 2015-03-07 Impact factor: 2.668