Novel eicosapentaenoic acid-derived F3-isoprostanes as biomarkers of lipid peroxidation

Abstract

Isoprostanes (iPs) are prostaglandin (PG) isomers generated by free radical-catalyzed peroxidation of polyunsaturated fatty acids (PUFAs). Urinary F(2)-iPs, PGF(2alpha) isomers derived from arachidonic acid (AA) are used as indices of lipid peroxidation in vivo. We now report the characterization of two major F(3)-iPs, 5-epi-8,12-iso-iPF(3alpha)-VI and 8,12-iso-iPF(3alpha)-VI, derived from the omega-3 fatty acid, eicosapentaenoic acid (EPA). Although the potential therapeutic benefits of EPA receive much attention, a shift toward a diet rich in omega-3 PUFAs may also predispose to enhanced lipid peroxidation. Urinary 5-epi-8,12-iso-iPF(3alpha)-VI and 8,12-iso-iPF(3alpha)-VI are highly correlated and unaltered by cyclooxygenase inhibition in humans. Fish oil dose-dependently elevates urinary F(3)-iPs in mice and a shift in dietary omega-3/omega-6 PUFAs is reflected by an increasing slope [m] of the line relating urinary 8, 12-iso-iPF(3alpha)-VI and 8,12-iso-iPF(2alpha)-VI. Administration of bacterial lipopolysaccharide evokes a reversible increase in both urinary 8,12-iso-iPF(3alpha)-VI and 8,12-iso-iPF(2alpha)-VI in humans on an ad lib diet. However, while excretion of the iPs is highly correlated (R(2) median = 0.8), [m] varies by an order of magnitude, reflecting marked inter-individual variability in the relative peroxidation of omega-3 versus omega-6 substrates. Clustered analysis of F(2)- and F(3)-iPs refines assessment of the oxidant stress response to an inflammatory stimulus in vivo by integrating variability in dietary intake of omega-3/omega-6 PUFAs.

Figures

FIGURE 1.

F3-iPs derived from EPA. A, formation and metabolism of isoprostanes. AA is more abundant than EPA and DHA in cell membranes obtained from individuals consuming a Western diet. Isoprostanes are formed from the corresponding PUFA substrate in situ following free radical attack. F2-iPs and F3-iPs are excreted into urine in their original form, but F4-iPs are at least partly metabolized to F3-iPs before excretion. (F2-iPs: F2-isoprostanes; F3-iPs: F3-isoprostanes; F4-iPs: F4-isoprostanes). B, six types of F3-iPs. C, 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI.

FIGURE 2.

Chromatograms of 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI in human and mouse urine. Representative selected reaction monitoring chromatograms of d4-5-epi-8,12-iso-iPF3α-VI and d4-8,12-iso-iPF3α-VI (A, panel a and B, panel a), and co-eluting peaks corresponding to the endogenous compound in human urine (A, panel b) and in mouse urine (B, panel b). Co-injection of exogenous synthetic 8,12-iso-iPF3α-VI (1 ng of standard to 1 ml of human urine shown in A, panel c and 0.5 ng of standard to 0.5 ml of mouse urine shown in B, panel c) and 5-epi-8,12-iso-iPF3α-VI (1 ng of standard to 1 ml of human urine shown in A, panel d and 0.5 ng of standard to 0.5 ml of mouse urine shown in B, panel d) results in increased peak height, but not width, indicating that these peaks are homogeneous (A, panels c and d and B, panels c and d). Transitions characteristic of F3-iPs are m/z 355→115 for d4-5-epi-8,12-iso-iPF3α-VI and d4-8,12-iso-iPF3α-VI and m/z 351→115 for 5-epi-8,12-iso-iPF3α-VI and 8,12-iso-iPF3α-VI.

FIGURE 3.

CID analysis. Product ion spectrum of d4-5-epi-8,12-iso-iPF3α-VI (A) and d4-8,12-iso-iPF3α-VI (C) with m/z values of 115, 177, 221, 275, 311, 337 and a base peak of 355. Product ion spectrum of the endogenous 5-epi-8,12-iso-iPF3α-VI (B) and 8,12-iso-iPF3α-VI (D) with m/z values of 115, 173, 217, 271, 307, 333 and a base peak of 351. The differences in m/z values between these two groups were either 0 or 4 mass units reflecting fragments with or without deuterium.

FIGURE 4.

Bioactivity of F2versus F3-iPs. A, intravenous injection of iPF2α-III (50 μg/kg body weight) caused a rapid and substantial increase in MAP in mice. B, intravenous injection of iPF3α-III (50 μg/kg body weight) failed to elevate MAP significantly. C, dose-dependent increase in MAP by iPF2α-III but not by iPF3α-III. D and E, pretreatment with iPF2α-III, but not iPF3α-III augments ADP-induced platelet aggregation.

FIGURE 5.

Biosynthesis of F3-iPs in mice fed with fish oil. A, no significant change in levels of 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI in mice on a high fat diet with 0% EPA (n = 5). B, in mice fed with a diet of 1% fish oil, urinary 8,12-iso-iPF3α-VI tended to increase but did not attain significance. A decline in urinary 8,12-iso-iPF2α-VI did attain significance. Five mice were studied in each group. C, in mice fed with a diet containing 5% fish oil, urinary 8,12-iso-iPF3α-VI significantly increased at 6 and 11 weeks. Urinary 8,12-iso-iPF2α-VI levels also significantly increased. Five mice were studied in each group. D and E, fish oil administration dose-dependently elevates urinary F3-iPs, reflected by an increasing slope of the line relating urinary 8,12-iso-iPF3α-VI and 8,12-iso-iPF2α-VI. (11 weeks on special diet). *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 6.

Biosynthesis of F3-iPs in humans. A, LPS (3 ng/kg) induces increased formation of F3-iPs. (n = 10). B, correlation of 8,12-iso-iPF3α-VI with 8,12-iso-iPF2α-VI in the group as a whole. C, individual correlations of 8,12-iso-iPF3α-VI with 8,12-iso-iPF2α-VI, among individuals.