A highly sensitive method for analysis of 7-dehydrocholesterol for the study of Smith-Lemli-Opitz syndrome

被引:40
|
作者
Liu, Wei [1 ]
Xu, Libin [1 ]
Lamberson, Connor [1 ]
Haas, Dorothea [3 ]
Korade, Zeljka [2 ]
Porter, Ned A. [1 ]
机构
[1] Vanderbilt Univ, Dept Chem, Vanderbilt Inst Chem Biol, Nashville, TN 37235 USA
[2] Vanderbilt Univ, Dept Psychiat, Vanderbilt Kennedy Ctr Res Human Dev, Nashville, TN 37235 USA
[3] Univ Childrens Hosp, Div Inborn Metab Dis, D-69120 Heidelberg, Germany
基金
美国国家科学基金会; 美国国家卫生研究院;
关键词
desmosterol; Diels-Alder cycloaddition; PTAD; liquid chromatography-mass spectrometry; gas chromatography; 7-dehydrocholesterol reductase; CHROMATOGRAPHY-MASS-SPECTROMETRY; SOLVENT-DEPENDENT CHANGES; ENE REACTION; CHOLESTEROL-BIOSYNTHESIS; MALFORMATION SYNDROMES; BIOLOGICAL-ACTIVITIES; CYSTIC-FIBROSIS; HUMAN PLASMA; MOUSE MODEL; RAT MODEL;
D O I
10.1194/jlr.D043877
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:329 / 337
页数:9
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