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Molecular & cellular toxicology v.3 no.4, 2007년, pp.273 - 281   피인용횟수: 1
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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

Lee, Youn-Sun   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Choi, Heon-Kyo   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Yoo, Jae-Myung   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Choi, Kyong-Mi   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Lee, Yong-Moon   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Oh, Sei-Kwan   (Department of Neuroscience, College of Medicine, Ewha Womans UniversityUU0001056  ); Kim, Tack-Joong   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Yun, Yeo-Pyo   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Hong, Jin-Tae   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  ); Okino, Nozomu   (Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityUU0014959  ); Ito, Makoto   (Department of Bioscience and Biotechnology, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu UniversityUU0014959  ); Yoo, Hwan-Soo   (College of Pharmacy and CBITRC, Chungbuk National UniversityUU0001309  );
  • 초록

    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-acidfree 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 BSAceramide 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.


  • 주제어

    Ceramide .   HPLC .   Cell death .   ICR mice .   LLCPK1 cells.  

  • 참고문헌 (22)

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  • 이 논문을 인용한 문헌 (1)

    1. 2008. "" Biomolecules & therapeutics, 16(2): 100~105     

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