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N-oleoyl-D-erythro-sphingosine-based analysis of ceramide by high performance liquid chromatography and its application to determination in diverse biological samples

Title
N-oleoyl-D-erythro-sphingosine-based analysis of ceramide by high performance liquid chromatography and its application to determination in diverse biological samples
Authors
Lee, Youn-SunChoi, Heon-KyoYoo, Jae-MyungChoi, Kyong-MiLee, Yong-MoonOh, SeikwanKim, Tack-JoongYun, Yeo-PyoHong, Jin-TaeOkino, NozomuIto, MakotoYoo, Hwan-Soo
Ewha Authors
오세관
SCOPUS Author ID
오세관scopus
Issue Date
2007
Journal Title
MOLECULAR & CELLULAR TOXICOLOGY
ISSN
1738-642XJCR Link
Citation
vol. 3, no. 4, pp. 273 - 281
Keywords
ceramideHPLCcell deathICR miceLLC-PK1 cells
Publisher
KOREAN SOC TOXICOGENOMICS & TOXICOPROTEOMICS
Indexed
SCIE; SCOPUS; KCI WOS
Abstract
Ceramide is involved in cell death as a lipid mediator of stress responses. In this study, we developed an improved method of ceramide quantification based on added synthetic ceramide and thin layer chromatography (TLC) separation, and applied to biological samples. Lipids were extracted from samples spiked with N-oleoyl-D-erythro-sphingosine (C-17 ceramide) as an internal standard. Ceramide was resolved by TLC, complexed with fatty-acid-free bovine serum albumin (BSA), and deacylated by ceramidase (CDase). The released sphingosine was derivatized with o-phthalaldehyde (OPA) and measured by high performance liquid chromatography (HPLC). The limit of detection for ceramide was about 1-2 pmol and the lower limit of quantification was 5 pmol. Ceramide recovery was approximately 86-93%. Ceramide concentrations were determined in biological samples including cultured cells, mouse tissues, and mouse and human plasma. TLC separation of ceramide provides HPLC chromatogram with a clean background without any interfering peaks and the enhanced solubility of ceramide by BSA-ceramide complex leads to the increased deacylation of ceramide. The use of an internal standard for the determination of ceramide concentration in these samples provides an accurate and reproducible analytical method, and this method can be applicable to diverse biological samples.
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의과대학 > 의학과 > Journal papers
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