Abstract
A method for separating, detecting, and quantifying cholesterol hydroperoxide (Ch-OOH) based on extraction, purification by solid-phase extraction cartridge, high-performance liquid chromatography with chemiluminescent detection (HPIC-CI), and liquid chromatography mass spectrometry has been developed for human erythrocyte membrane. We prepared standard compounds of the cholesterol 5α-, 7α-, and 7β-hydroperoxides (Ch 5α-OOH, Ch 7α-OOH, and Ch 7β-OOH). An octyl silica column with methanol/water/acetonitrile 89∶9∶2 (by vol) as eluent was used to determine Ch-OOH. HPLC-CL that incorporated cytochrome c and luminol as the post-column luminescent reagent was used. We also investigated the optimal assay conditions and how to prevent formation of artifact Ch-OOH. Analysis of erythrocyte membranes from seven healthy volunteers identified Ch 7α-OOH and Ch 7β-OOH, but not Ch 5α-OOH, as commonly occurring components. The respective mean concentrations of Ch 7α-OOH and Ch 7β-OOH were 2,5±1.6 and 5A±3.5 pmol/mL blood.
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Abbreviations
- 7α-OH:
-
7α-hydroxycholesterol
- 7β-OH:
-
7β-hydroxycholesterol
- BITT:
-
5-di-tert-buty-4-hydroxytoluene
- Ch-OOH:
-
cholesterol hydroperoxides
- Ch 5α-OOH:
-
cholesterol 5α-hydroperoxide
- Ch 7α-OOH:
-
cholesterol 7α-hydroperoxide
- Ch 7β-OOH:
-
cholesterol 7β-hydroperoxide
- CI:
-
chomiluminescent, chemiluminescence
- DMF:
-
dimethylfuran
- HPLC-CI:
-
high-performance liquid chromatography with chemiluminescent detection
- IS:
-
internal standard
- LC-MS:
-
liquid chromatography-mass spaectrometry
- NMR:
-
nuclear magnetic resonance
- PCOOH:
-
phosphatidylcholine hydroperoxide
- UV:
-
ultraviolet
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Adachi, J., Asano, M., Naito, T. et al. Chemiluminescent determination of cholesterol hydroperoxides in human erythrocyte membrane. Lipids 33, 1235–1240 (1998). https://doi.org/10.1007/s11745-998-0329-0
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DOI: https://doi.org/10.1007/s11745-998-0329-0