HPLC-FLD determination of NBD-cholesterol, its ester and other metabolites in cellular lipid extracts

Our plain-English summary of HPLC-FLD determination of NBD-cholesterol, its ester and other metabolites in cellular lipid extracts, by X.Z. Cao, T.Y. Mi, L. Li, et al (Biomedical Chromatography, 2013).

The study

• Authors: X.Z. Cao, T.Y. Mi, L. Li, M.A. Vermeer, C.C. Zhang, N. Huang, J.K. Manoj
• Journal: Biomedical Chromatography
• Published: 2013;27(7):910-5
• Cited by: 5 articles, as counted by NIH iCite
• Read it: PubMed · Publisher

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.

Copyright © 2013 John Wiley & Sons, Ltd.

In plain English

This laboratory study did not examine asbestos or any asbestos-related disease. It described a chemical method for measuring a fluorescent cholesterol tracer, NBD-cholesterol, inside cultured cells. The authors developed and validated a liquid chromatography method with fluorescence detection. They tested it in Caco-2 human colon adenocarcinoma cells and in AC29 mouse malignant mesothelioma cells engineered to carry the cholesterol-processing enzyme genes acat1 or acat2. The method showed no interference from cell material, a detection limit of 30 ng/mL, and recoveries of 81.7% and 90.6% at two test concentrations. Besides the tracer and its oleate ester, Caco-2 cells held two other tracer products. Based on mass spectrometry, the authors judged these likely to be an oxidation product and an isomer. The AC29 cells showed only one, matching the oxidation product. In both AC29 cell lines, the known enzyme inhibitor YIC-C8-434 significantly lowered the ester level in a dose-dependent way and raised free tracer levels. The authors concluded the method was sensitive and specific for analyzing NBD-cholesterol uptake, esterification and metabolism in cultured cells.

We wrote this plain-English summary from the full article with the help of AI, and a second AI model checked each sentence against the article. It is not the authors' wording; the abstract and the article are their own account.

What the study did and found

• Design: Laboratory method-development and validation study in cultured cells (no human subjects, no asbestos exposure)
• Who was studied: Cultured cell lines: Caco-2 human colon adenocarcinoma cells, and AC29 mouse malignant mesothelioma cells stably transfected with the acat1 gene (AC29/ACAT1) or the acat2 gene (AC29/ACAT2). The AC29 cells were supplied by a researcher at Wake Forest University. The work was done at Unilever R&D Shanghai and collaborating sites.
• Exposure: Cells were incubated with the fluorescent cholesterol analog NBD-cholesterol, with or without different concentrations of the ACAT inhibitor YIC-C8-434, for 4 hours. Some Caco-2 incubations ran for 24 hours. No asbestos or fibers were studied.
• Main results:
  • Lipid extracts from all three cell lines without NBD-cholesterol gave no significant fluorescent signal, which the authors took to mean there was no interference from the cell material.
  • The limit of detection for NBD-cholesterol was 30 ng/mL.
  • Recovery of NBD-cholesterol from Caco-2 cell extracts was 81.7% and 90.6% at the two tested concentrations. Within-batch variation (RSD) was under 9.7% and between-batch variation was under 2.8%.
  • In Caco-2 cell extracts, two extra peaks (M1-1 and M1-2) were seen besides NBD-cholesterol and its oleate ester. By mass spectrometry, M1-1 had a molecular weight of 492.4, two units lower than NBD-cholesterol, and M1-2 had the same molecular weight as NBD-cholesterol.
  • In AC29/ACAT1 and AC29/ACAT2 cells, only one additional metabolite was detected in each. It showed the same mass spectrometry fragmentation pattern as M1-1.
  • NBD-cholesteryl oleate was only at trace levels in Caco-2 cells, so quantification was not done in Caco-2 cells.
  • In AC29/ACAT1 and AC29/ACAT2 cells (n = 4), YIC-C8-434 significantly decreased NBD-cholesteryl oleate in a dose-dependent way (p less than 0.05) and significantly increased intact NBD-cholesterol (p less than 0.05). In AC29/ACAT1 cells, metabolite M2-1 was mildly but significantly reduced at the two higher doses. In AC29/ACAT2 cells, M3-1 showed a mild decreasing trend that was not statistically significant.
• The authors' conclusion: The authors concluded that a sensitive and specific HPLC-FLD method was established and validated for analyzing NBD-cholesterol uptake, esterification and metabolism in cultured cells.
• Limitations the authors state:
  • The NBD-cholesteryl oleate signal was low in Caco-2 cells even after 24 hours. The authors said this batch of Caco-2 cells may not be an appropriate model for assessing cholesterol esterification.
  • The structures of the additional metabolites were inferred rather than confirmed. The authors described them as speculated.

Topics

Medical terms in this study, explained in our Lexicon: Adenocarcinoma, Malignant Mesothelioma.

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