Abstract
Esterified cholesterol content is often lower than free cholesterol content in biological systems and thus the determination of the esterified cholesterol content of cells is often challenging. Traditional methods use enzymatic assays in which an indirect measurement of the esterified cholesterol content is obtained by subtracting the measurements of the free from the total cholesterol content. However, this approach fails in the case where the total cholesterol content of cells is unchanged while the ratio of free to esterified cholesterol changes such that total and free cholesterol content are very similar and thus the difference may fall within the background noise of the enzymatic assay. To overcome this challenge, we here describe a method that utilizes a TLC-based technique to isolate esterified cholesterol. Isolated esterified cholesterol can then be measured using traditional enzymatic methods. Therefore, this method provides a practical and more sensitive assay to measure esterified cholesterol content in cellular extracts.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Mendez AJ (1997) Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion. J Lipid Res 38(9):1807–1821
Liebisch G, Binder M, Schifferer R, Langmann T, Schulz B, Schmitz G (2006) High throughput quantification of cholesterol and cholesteryl ester by electrospray ionization tandem mass spectrometry (ESI-MS/MS). Biochim Biophys Acta 1761(1):121–128. doi:10.1016/j.bbalip.2005.12.007
Amundson DM, Zhou M (1999) Fluorometric method for the enzymatic determination of cholesterol. J Biochem Biophys Methods 38(1):43–52
De Hoff JL, Davidson LM, Kritchevsky D (1978) An enzymatic assay for determining free and total cholesterol in tissue. Clin Chem 24(3):433–435
Smith RM, Marble A (1937) The colorimetric determination of free and combined cholesterol. J Biol Chem 117:673–684
DeBarber AE, Lutjohann D, Merkens L, Steiner RD (2008) Liquid chromatography-tandem mass spectrometry determination of plasma 24S-hydroxycholesterol with chromatographic separation of 25-hydroxycholesterol. Anal Biochem 381(1):151–153. doi:10.1016/j.ab.2008.05.037
Zak B (1977) Cholesterol methodologies: a review. Clin Chem 23(7):1201–1214
Li L, Han J, Wang Z, Liu J, Wei J, Xiong S, Zhao Z (2014) Mass spectrometry methodology in lipid analysis. Int J Mol Sci 15(6):10492–10507. doi:10.3390/ijms150610492
Acknowledgments
This work was supported by the NIH and NIDDK (grant numbers DK090316, DK104753) and by Grant Number 1UL1TR000460, University of Miami Clinical and Translational Science Institute, from the National Center for Advancing Translational Sciences and the National Institute on Minority Health and Health Disparities.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2017 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Pedigo, C.E., Merscher, S.M., Fornoni, A. (2017). Direct Measurement of Free and Esterified Cholesterol Mass in Differentiated Human Podocytes: A TLC and Enzymatic Assay-Based Method. In: Bhattacharya, S. (eds) Lipidomics. Methods in Molecular Biology, vol 1609. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6996-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-6996-8_6
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-6995-1
Online ISBN: 978-1-4939-6996-8
eBook Packages: Springer Protocols