title: HPLC-FLD determination of NBD-cholesterol, its ester and other metabolites in cellular lipid extracts.
abstract: 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 cholesterol tracer used to study cholesterol absorption and metabolism in cultured cells. In order to measure free intracellular cholesterol and its esters, a precise and sensitive method employing high-performance liquid chromatography/fluorescence detection (HPLC-FLD) was developed for the first time. Method validation showed a limit of detection at 30 ng/mL. The calibration curve was linear within the range of 0.0625-10.0 µg/mL (r(2) = 0.999). Accuracy and precision were highlighted by good recovery and low variations. Apart from NBD-cholesteryl oleate, two additional cellular metabolites of NBD-cholesterol, probably an isomer and an oxidation product, were determined in the lipid extracts of Caco-2 human colon adenocarcinoma cells according to mass spectrometry. In AC29 mouse malignant mesothelioma cells overexpressing acyl-CoA:cholesterol acyltransferase-1 (ACAT1) or ACAT2, only the oxidized metabolite was detected. Using the newly developed method, YIC-C8-434, a known ACAT inhibitor, was shown to inhibit ACAT activity in Caco-2 cells, as well as in AC29/ACAT1 or AC29/ACAT2 cells. In conclusion, the sensitive and specific HPLC-FLD method is a powerful tool for simultaneous quantification of intracellular NBD-cholesterol and its oleoyl-ester.
authors.affiliation: Unilever R&D Shanghai, 66 Lin Xin Road, Shanghai, 200335, People's Republic of China.
authors.forename: Xiu-zhen
authors.initials: XZ
authors.lastname: Cao
authors.forename: Ting-yan
authors.initials: TY
authors.lastname: Mi
authors.forename: Lin
authors.initials: L
authors.lastname: Li
authors.forename: Mario A
authors.initials: MA
authors.lastname: Vermeer
authors.forename: Chen-chen
authors.initials: CC
authors.lastname: Zhang
authors.forename: Nan
authors.initials: N
authors.lastname: Huang
authors.forename: Joshi K
authors.initials: JK
authors.lastname: Manoj
mesh_terms: D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004952:Esters / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D042541:Intracellular Space / Q000737:chemistry / Q000378:metabolism; D016014:Linear Models; D013058:Mass Spectrometry; D051379:Mice; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods*
chemical_list: C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D004952:Esters; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan