Oxidized derivatives of ω-3 fatty acids:: identification of IPF3α-VI in human urine

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 omega 3-PUFAs have been found to be below detection limits of available assays. Because of the interest in omega 3-PUFA dietary supplementation, we developed specific methods to measure nPF(4 alpha)-VI and iPF(3 alpha)-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), and liquid chromatography tandem mass spectrometry (LC/MS/MS). Although nPF(4 alpha)-VI was below the detection limit of the assay, we conclusively identified iPF(3 alpha)-VI in human urine by GC/MS and LC/MS/MS. The mean levels in 26 subjects were similar to 300 pg/mg creatinine. Our failure to detect nPF(4 alpha)-VI may have been due to its rapid metabolism by beta-oxidation to iPF(3 alpha)-VI, which we showed to occur in rat liver homogenates. In contrast, iPF(3 alpha)-VI is highly resistant to beta- oxidation in vitro. Thus iPF(3 alpha)-VI can be formed by two mechanisms: i) direct autoxidation of EPA, and ii) b- oxidation of nPF(4 alpha)-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.