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Journal of Lipid Research


Isoprostanes (iPs) are prostaglandin-like molecules derived from autoxidation of polyunsaturated fatty acids (PUFAs). Urinary iP levels have been used as indices of in vivo lipid peroxidation. Thus far, it has only been possible to measure iPs derived from arachidonic acid in urine, because levels of iPs/neuroprostanes (nPs) derived from ω3-PUFAs have been found to be below detection limits of available assays. Because of the interest in ω3-PUFA dietary supplementation, we developed specific methods to measure nPF4α-VI and iPF3α-VI [derived from 4,7,10,13,16,19-docosahexaenoic acid (DHA) and 5,8,11,14,17-eicosapentaenoic acid (EPA)] using a combination of chemical synthesis, gas chromatography/mass spectrometry (GC/MS), andliquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF4α-VI was below the detection limit of the assay, we conclusively identified iPF3α-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were ∼300 pg/mg creatinine. Our failure to detect nPF4α-VI may have been due to its rapid metabolism by β-oxidation to iPF3α-VI, which we showed to occur in rat liver homogenates. In contrast, iPF3α- VI is highly resistant to β-oxidation in vitro. Thus iPF 3α-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) β-oxidation of nPF4α-VI, formed by autoxidation of DHA. This iP may therefore serve as an excellent marker for the combined in vivo peroxidation of EPA and DHA.

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