A reliable tool for detecting 7‐dehydrocholesterol and cholesterol in human plasma and its use in diagnosis of Smith–Lemli–Opitz syndrome

作者：Yitao Luo, Zhengyuan Liu, Yujie Zeng, Yuxiao Zhang, Yujing Luan, Li Ma, Li Chen, Lin Zou, H. J. Yang, Zhibin Huang, Yulan Rao, Chengqiang Zhang · 发表于：Journal of Separation Science · 年份：2021 · DOI：10.1002/jssc.202100594 · 被引用次数：7 · 研究领域：Cholesterol and Lipid Metabolism、Peroxisome Proliferator-Activated Receptors、Metabolism and Genetic Disorders

BACKGROUND: Smith-Lemli-Opitz syndrome is a birth defect caused by the deficiency of 7-dehydrocholesterol reductase in cholesterol biosynthesis pathway, which leads to accumulation of 7-dehydrocholesterol and reduction of cholesterol in body fluids. To effectively diagnose Smith-Lemli-Opitz syndrome and monitor therapy, a reliable method for simultaneous detection of 7-dehydrocholesterol and cholesterol is needed. METHODS: In the presence of antioxidants (2,6-ditert-butyl-4-methylphenol and triphenylphosphine), 50 μL of human plasma were hydrolyzed at 70℃ for 40 min with 1 M potassium hydroxide in 90% ethanol, and then 7-dehydrocholesterol and cholesterol were extracted by 600 μL of n-hexane for three times. After microwave-assisted derivatization with 70 μL of N,O-bis(trimethylsilyl)trifluoroacetamide at 460 W for 3 min, the analytes were measured by gas chromatography-mass spectrometry. RESULTS: The limits of detection were 100 ng/mL for 7-dehydrocholesterol and 300 ng/mL for cholesterol. Good linearity was obtained in the range of 1-600 μg/mL for 7-dehydrocholesterol and 10-600 μg/mL for cholesterol, which completely covered the biochemical levels of Smith-Lemli-Opitz syndrome patients that have been reported. CONCLUSION: A time-saving and accurate gas chromatography with mass spectrometry based method was developed for the determination of 7-dehydrocholesterol and cholesterol in human plasma, which also serves as a useful tool for Smith-Lemli-Opitz syndrome diagnosis, tre...