Measurement of multiple oxidized lipid products from a single plasma sample from healthy volunteers and clinical patients

Abstract

Many oxidized lipid products have been associated with diseases such as atherosclerosis, stroke, thalassemia and diabetes mellitus. These products include F2-isoprostanes (F2-IsoPs), hydroxyeicosatetraenoic acid products (HETEs) and oxysterols or cholesterol oxidation products (COPs). Each product is often measured individually in separate blood samples. Here we present a method capable of measuring F2-IsoPs, HETEs, COPs and arachidonate using a single plasma or serum sample. As examples for the application of the method, clinical patient data from stroke, dengue and diabetic mellitus patients are presented. A volume of plasma or serum sample (1 ml) is spiked with heavy isotopic standards and hydrolysed in alkali with organic solvent. It is then purified using anionic exchange solid phase extraction (SPE). After washing the SPE column, hexane and hexane/ethyl acetate portions are collected and combined for COPs measurement. Afterwards, the column is loaded with hexane/ethanol/acetic acid and fractions collected for total F2-IsoPs, total HETEs and arachidonate measurement. All compounds in the eluates are measured by gas chromatography-mass spectrometry. The robustness of the method was tested by the efficiency of SPE and reproducibility for all compounds measured. The method showed high and reliable efficiency of SPE and reproducibility of oxidized lipid products and were measurable in plasma of healthy volunteers and clinical patients. For example, levels of total F2- IsoPs 0.4590.26 ng/ml (n 157), total HETEs 34.1916.4 ng/ml (n 21) total arachidonate 68.4924.5 ug/ml (n 33), and COPs 7- ketocholesterol 12.396.6 ng/ml, 7b-hydroxycholesterol 6.393.5 ng/ ml, 7a-hydroxycholesterol 15.197.1 ng/ml, 24-hydroxycholesterol 41.4918.2 ng/ml and 27-hydroxycholesterol 29.1916.8ng/ml (n 26) were recorded in healthy volunteers. The method was also applicable in stroke patients where total F2-IsoPs 0.9890.38 ng/ml (n 21), total HETEs 33.9922.1 ng/ml (n 15), total arachidonate 90.4930.7 ug/ml (n 21), and COPs 7-ketocholesterol 27.9917.6 ng/ml, 7b-hydroxycholesterol 31.5918.9 ng/ml, 7a-hydroxycholesterol 5.591.6 ng/ml, 24-hydroxycholesterol 124.69102.4 ng/ml and 27- hydroxycholesterol 37.6919.0 ng/ml (n 21) were recorded. Our investigation presented indicates that the method developed is applicable for the measurement of lipid oxidation products in human clinical settings or in small animals where plasma samples are limited.