HPLC-FLD determination of NBD-cholesterol, its ester and other metabolites in cellular lipid extracts.
作者： Xiu-zhen Cao , Ting-yan Mi , Lin Li , Mario A. Vermeer , Chen-chen Zhang
DOI: 10.1002/BMC.2881
关键词: Chemistry 、 Cholesterol 、 Sterol O-acyltransferase 、 Metabolism 、 Caco-2 、 Cholesterol analog 、 Chromatography 、 Metabolite 、 Detection limit 、 Biochemistry 、 Intracellular
摘要: 22-[N(-7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-23,24-bisnor-5-cholen-3β-ol (NBD-cholesterol), a fluorescent cholesterol analog, was an extragenous tracer used to study absorption and metabolism in cultured cells. In order measure free intracellular its esters, precise sensitive method employing high-performance liquid chromatography/fluorescence detection (HPLC-FLD) developed for the first time. Method validation showed limit of at 30 ng/mL. The calibration curve linear within range 0.0625-10.0 µg/mL (r(2) = 0.999). Accuracy precision were highlighted by good recovery low variations. Apart from NBD-cholesteryl oleate, two additional cellular metabolites NBD-cholesterol, probably isomer oxidation product, determined lipid extracts Caco-2 human colon adenocarcinoma cells according mass spectrometry. AC29 mouse malignant mesothelioma overexpressing acyl-CoA:cholesterol acyltransferase-1 (ACAT1) or ACAT2, only oxidized metabolite detected. Using newly method, YIC-C8-434, known ACAT inhibitor, shown inhibit activity cells, as well AC29/ACAT1 AC29/ACAT2 conclusion, specific HPLC-FLD is powerful tool simultaneous quantification NBD-cholesterol oleoyl-ester.
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