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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.




The following articles in journals at HighWire Press have cited this article:


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J. F. Collins, Z. Hu, P. N. Ranganathan, D. Feng, L. M. Garrick, M. D. Garrick, and R. W. Browne
Induction of arachidonate 12-lipoxygenase (Alox15) in intestine of iron-deficient rats correlates with the production of biologically active lipid mediators
Am J Physiol Gastrointest Liver Physiol, April 1, 2008; 294(4): G948 - G962.
[Abstract] [Full Text] [PDF]

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 Hum ReprodHome page
L.W. Jackson, E.F. Schisterman, R. Dey-Rao, R. Browne, and D. Armstrong
Oxidative stress and endometriosis
Hum. Reprod., July 1, 2005; 20(7): 2014 - 2020.
[Abstract] [Full Text] [PDF]


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