Femtomole analysis of 9-oxononanoyl cholesterol by high performance liquid chromatography1

Journal of Lipid Research

Wolfgang Sattler¹  Peter M. Abuja¹  Herbert Boechzelt²  Karl Oettl²  Barbara Karten³  Martin Mittelbach⁴ 

[1] 8010 Graz, Austria;University of Graz;Institute of Biochemistry, SFB Biomembranes Research Centre;Institute of Organic Chemistry, SFB Biomembranes Research Centre;

关键词: LDL;    atherosclerosis;    plaque lipids;    reactive aldehydes;    lipid peroxidation;    core-aldehydes;   

DOI  :  

来源: DOAJ

【 摘 要 】

9-Oxononanoyl cholesterol, a cholesterol core-aldehyde formed during lipoprotein oxidation, was recently identified in advanced human atherosclerotic lesions. Here we present a rapid and sensitive HPLC method for 9-oxononanoyl cholesterol analysis. 9-Oxononanoyl cholesterol was converted to the corresponding fluorescent decahydroacridine derivative by reaction with 1,3-cyclohexanedione. The derivatives formed were purified by solid-phase extraction on C-18 columns, separated by reversed phase HPLC with isocratic elution, and detected by their fluorescence. Decahydroacridine derivatives of 9-oxononanoyl cholesterol were stable for at least 160 h. The limit of quantitation of the method presented is at the low (≈50) femtomole level, with an absolute limit of detection (signal: noise = 6) of 15 fmol. Intra-assay variation was ≤5%, while inter-assay variations were between 5 and 15%, depending on the concentration of the analyte. Standard curves were linear over nearly three orders of magnitude (50 fmol–12.5 pmol). 9-Oxononanoyl cholesterol proved to be the major cholesterol core-aldehyde formed during t-BuOOH/FeSO4 oxidation of cholesteryl linoleate and Cu2+-induced LDL oxidation, findings confirmed by atmospheric pressure chemical ionization–mass spectrometry. Analysis of lipid extracts obtained from advanced human atherosclerotic lesions revealed the presence of 9-oxononanoyl cholesterol in all tissue samples analyzed (28 ± 14 μmol/mol cholesterol, n = 9) despite the presence of α-tocopherol (4 ± 1.2 mmol/mol cholesterol, n = 9).—Karten, B., H. Boechzelt, P. M. Abuja, M. Mittelbach, K. Oettl, and W. Sattler. Femtomole analysis of 9-oxononanoyl by high performance liquid chromatography. J. Lipid Res. 1998. 39: 1508–1519.

【 授权许可】

Unknown