Premium
Chemiluminescent determination of cholesterol hydroperoxides in human erythrocyte membrane
Author(s) -
Adachi Junko,
Asano Migiwa,
Naito Takeaki,
Ueno Yasuhiro,
Tatsuno Yoshitsugu
Publication year - 1998
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-998-0329-0
Subject(s) - chemistry , chromatography , chemiluminescence , luminol , high performance liquid chromatography , reagent , column chromatography , extraction (chemistry) , methanol , solid phase extraction , organic chemistry
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 5 A ±3.5 pmol/mL blood.