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Clinical Chemistry 46: 829-836, 2000;
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(Clinical Chemistry. 2000;46:829-836.)
© 2000 American Association for Clinical Chemistry, Inc.

Articles

HPLC Analysis of Lipid-derived Polyunsaturated Fatty Acid Peroxidation Products in Oxidatively Modified Human Plasma

Richard W. Browne1,2,a and Donald Armstrong1

Departments of
1 Clinical Laboratory Science and
2 Social and Preventive Medicine, State University of New York at Buffalo, Buffalo, NY 14214.
a Address correspondence to this author at: Department of Social and Preventive Medicine, State University of New York at Buffalo, 270 Farber Hall, 3435 Main St., Buffalo, NY 14214. Fax 716-829-2979; e-mail rwbrowne{at}acsu.buffalo.edu

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.






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