title: A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome.
abstract: We describe a highly sensitive method for the detection of 7-dehydrocholesterol (7-DHC), the biosynthetic precursor of cholesterol, based on its reactivity with 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) in a Diels-Alder cycloaddition reaction. Samples of biological tissues and fluids with added deuterium-labeled internal standards were derivatized with PTAD and analyzed by LC-MS. This protocol permits fast processing of samples, short chromatography times, and high sensitivity. We applied this method to the analysis of cells, blood, and tissues from several sources, including human plasma. Another innovative aspect of this study is that it provides a reliable and highly reproducible measurement of 7-DHC in 7-dehydrocholesterol reductase (Dhcr7)-HET mouse (a model for Smith-Lemli-Opitz syndrome) samples, showing regional differences in the brain tissue. We found that the levels of 7-DHC are consistently higher in Dhcr7-HET mice than in controls, with the spinal cord and peripheral nerve showing the biggest differences. In addition to 7-DHC, sensitive analysis of desmosterol in tissues and blood was also accomplished with this PTAD method by assaying adducts formed from the PTAD "ene" reaction. The method reported here may provide a highly sensitive and high throughput way to identify at-risk populations having errors in cholesterol biosynthesis.
authors.affiliation: Department of Chemistry, Vanderbilt Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37235.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Libin
authors.initials: L
authors.lastname: Xu
authors.forename: Connor
authors.initials: C
authors.lastname: Lamberson
authors.forename: Dorothea
authors.initials: D
authors.lastname: Haas
authors.forename: Zeljka
authors.initials: Z
authors.lastname: Korade
authors.forename: Ned A
authors.initials: NA
authors.lastname: Porter
mesh_terms: D000818:Animals; D001774:Blood Chemical Analysis / Q000379:methods*; D045744:Cell Line, Tumor; D003684:Dehydrocholesterols / Q000097:blood* / Q000737:chemistry / Q000378:metabolism; D006579:Heterozygote; D006801:Humans; D051379:Mice; D009420:Nervous System / Q000378:metabolism; D019082:Smith-Lemli-Opitz Syndrome / Q000097:blood* / Q000235:genetics / Q000378:metabolism; D014230:Triazoles / Q000737:chemistry
chemical_list: D003684:Dehydrocholesterols; D014230:Triazoles; C053540:4-phenyl-1,2,4-triazoline-3,5-dione; C016705:7-dehydrocholesterol
keywords: 7-dehydrocholesterol reductase; Diels-Alder cycloaddition; PTAD; desmosterol; gas chromatography; liquid chromatography-mass spectrometry