Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells.
We developed a powerful gas chromatographic/mass spectrometric method allowing quantitative analysis of 11 structurally similar cholesterol precursors and plant sterols (squalene, desmosterol, 7-dehydrocholesterol, lathosterol, zymosterol, dihydro-lanosterol, lanosterol, FF-MAS, T-MAS, campesterol, sitosterol) from cultured human hepatocytes in a single chromatographic run. Deuterium labelled cholesterol, sitosterol and lathosterol were used as internal standards. Care was taken to select ions for the detection that gave the most appropriate discrimination in the assay. Replicate analyses gave a coefficient of variation less than 6%. Recovery experiments were satisfactory for 7-dehydrocholesterol, campesterol, desmosterol, lathosterol, zymosterol and cholesterol with less than 7% difference between expected and found levels. For other sterols, the difference between expected and found levels varied between 10 and 16%. It is concluded that this method is suitable for studies on the effect of different inhibitors and stimulators of cholesterol synthesis in cultured cells. Additionally, the method is relevant also for clinical applications since abnormally increased late cholesterol intermediates in patients are representations of the inherited disorders linked to different enzyme defects in the post-squalene cholesterol biosynthesis.
Acimovic
Jure
J
Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Lövgren-Sandblom
Anita
A
Monostory
Katalin
K
Rozman
Damjana
D
Golicnik
Marko
M
Lutjohann
Dieter
D
Björkhem
Ingemar
I
D000328:Adult; D002478:Cells, Cultured; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry / Q000379:methods*; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D008297:Male; D010840:Phytosterols / Q000737:chemistry*
D010840:Phytosterols; D002784:Cholesterol