Measurement of F2-isoprostanes, hydroxyeicosatetraenoic products, and oxysterols from a single plasma sample

Oxidized lipids such as F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs), and cholesterol oxidation products (COPs) are widely believed to be involved in multiple diseases. Usually, each product is measured individually in separate blood samples. In this study we describe a method allowing us to measure F2-IsoPs, HETEs, COPs, and arachidonate using a single sample. Plasma (1 ml) samples from healthy volunteers were diluted with heavy isotopic standards, hydrolyzed in alkali with organic solvent, and then subjected to anionic-exchange solid-phase extraction (SPE). After the SPE column was washed, hexane and hexane/ethyl acetate portions were collected and combined for COPs measurement. Thereafter the column was loaded with hexane/ethanol/acetic acid and fractions were collected for total F2-IsoPs, total HETEs, and arachidonate measurement. All compounds in the eluates were measured by gas chromatography–mass spectrometry. The efficiency of SPE and reproducibility for all compounds measured were high. Levels of total F2-IsoPs (0.45 ± 0.26 ng/ml (n = 157)), total HETEs (34.06 ± 16.35 ng/ml (n = 21)), total arachidonate (68.36 ± 24.45 μg/ml (n = 33)), and COPs (7-ketocholesterol, 12.25 ± 6.56 ng / ml; 7β-hydroxycholesterol, 6.32 ± 3.46 ng / ml; 7α-hydroxycholesterol, 15.06 ± 7.06 ng/ml; 24-hydroxycholesterol, 41.39 ± 18.22 ng/ml; and 27-hydroxycholesterol, 29.08 ± 16.79 ng/ml (n = 26)) were recorded in healthy subjects (age range 20 to 66 years; average male to female ratio 1:1).