Affiliation:
1. University Hospital Regensburg, Regensburg, Germany
2. Department of Psychiatry and Psychotherapy, University of Regensburg, Regensburg, Germany
Abstract
BACKGROUND
Cholesterol precursors and plant sterols have considerable potential as plasma biomarkers in several disorders of sterol metabolism and intestinal sterol absorption. Oxysterols are associated with atherogenesis, neurodegeneration, and inflammation. We developed a GC-MS method for the simultaneous analysis of these species in human plasma, including 24-, 25-, 27-hydroxycholesterol; 7-ketocholesterol; lanosterol; lathosterol; 7-dehydrocholesterol; desmosterol; stigmasterol; sitosterol; and campesterol.
METHODS
Sterols were hydrolyzed with ethanolic potassium hydroxide solution, extracted by liquid/liquid extraction with n-hexane, and derivatized with N-methyl-N-trimethylsilyl-trifluoracetamide. Positive chemical ionization with ammonia, as reagent gas, was applied to generate high abundant precursor ions.
RESULTS
The definition of highly sensitive precursor/product ion transitions, especially for coeluting substances, allowed fast gas chromatography run times of under 8.5 min. Using the multiple reaction monitoring mode, detection limits in the picogram per milliliter range could be achieved for most compounds. The method was validated for precision and recovery. Intraassay and interassay CVs were mostly <15% for serum and plasma samples. The recoveries of supplemented plasma samples in different concentrations were 88%–117%. The method was applied to stratification of patients with disorders in cholesterol biosynthesis and/or cholesterol absorption in hypercholesterolemia. The method revealed associations of sterol species with thyroid dysfunction and type 2 diabetes.
CONCLUSIONS
This method allows high-throughput sterol profiling in various diseases.
Funder
Actelion
European Community's Seventh Framework Programme
BMBF
Clinical Chemistry and Laboratory Medicine University Hospital Regensburg
Publisher
Oxford University Press (OUP)
Subject
Biochemistry (medical),Clinical Biochemistry
Cited by
59 articles.
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