HPLC analysis of lipid-derived polyunsaturated fatty acid peroxidation products in oxidatively modified human plasma

Background: Lipid peroxidation is a prominent manifestation of free radical activity and oxidative stress in biological systems. Diverse methodologies have been developed that measure a variety of lipid peroxidation products used as markers of lipid peroxidation processes. Methods: Hydroxy and hydroperoxy polyunsaturated fatty acid (PUFA) peroxidation products were analyzed in human blood plasma by reversed-phase HPLC after liquid-liquid extraction of total lipids and alkaline hydrolysis of lipid esters to liberate free PUFAs. An isocratic mobile phase containing 1 g/L acetic acid-acetonitrile-tetrahydrofuran (52:30:18, by volume) over 60 min duration, with ultraviolet absorbance detection at 236 nm by photodiode array, enabled the resolution and quantification of 13 regioisomeric hydroxy and hydroperoxy PUFAs. Results: As little as 250 μL of human plasma was utilized with an analytical range of 0.033-1.6 μmol/L for each compound. Intra- and interassay CVs for all compounds detected in normal or oxidatively modified human plasma were 3.2-11% and 4.7-12%, respectively. Analytical recoveries were 87-103%. Analysis of human plasma exposed to artificial oxidation with Cu²⁺ ion and hydrogen peroxide, a free radical-generating reaction, showed marked increases in hydroxy and hydroperoxy PUFA concentrations. Conclusion: Lipid-derived hydroxy and hydroperoxy PUFAs may be useful as clinical markers of lipid peroxidation and oxidative stress in the peripheral circulation. (C) 2000 American Association for Clinical Chemistry.