Formation of F-ring Isoprostane-like Compounds (F3-Isoprostanes) in Vivo from Eicosapentaenoic Acid

Abstract

Eicosapentaenoic acid (EPA, C20:5, -3) is the most abundant polyunsaturated fatty acid (PUFA) in fish oil. Recent studies suggest that the beneficial effects of fish oil are due, in part, to the generation of various free radical-generated non-enzymatic bioactive oxidation products from -3 PUFAs, although the specific molecular species responsible for these effects have not been identified. Our research group has previously reported that pro-inflammatory prostaglandin F 2 -like compounds, termed F 2 -isoprostanes (IsoPs), are produced in vivo by the free radical-catalyzed peroxidation of arachidonic acid and represent one of the major products resulting from the oxidation of this PUFA. Based on these observations, we questioned whether F 2 -IsoP-like compounds (F 3 -IsoPs) are formed from the oxidation of EPA in vivo. Oxidation of EPA in vitro yielded a series of compounds that were structurally established to be F 3 -IsoPs using a number of chemical and mass spectrometric approaches. The amounts formed were extremely large (up to 8.7 ؉ 1.0 g/mg EPA) and greater than levels of F 2 -IsoPs generated from arachidonic acid. We then examined the formation of F 3 -IsoPs in vivo in mice. Levels of F 3 -IsoPs in tissues such as heart are virtually undetectable at baseline, but supplementation of animals with EPA markedly increases quantities up to 27.4 ؉ 5.6 ng/g of heart. Interestingly, EPA supplementation also markedly reduced levels of pro-inflammatory arachidonate-derived F 2 -IsoPs by up to 64% ( p < 0.05). Our studies provide the first evidence that identify F 3 -IsoPs as novel oxidation products of EPA that are generated in vivo. Further understanding of the biological consequences of F 3 -IsoP formation may provide valuable insights into the cardioprotective mechanism of EPA.

Human Biochemistry of the Isoprostane Pathway

“…Interestingly, our laboratory has recently shown that the levels of IsoPs generated from the oxidation of EPA significantly exceed those of F 2 -IsoPs generated from arachidonic acid, perhaps because EPA contains more double bonds and is therefore more easily oxidizable (4). Additionally, in vivo in mice, levels of F 3 -IsoPs in tissues such as heart were virtually undetectable at base line, but supplementation of animals with EPA markedly increased quantities up to 27.4 Ϯ 5.6 ng/g of heart tissue.…”

“…F-ring IsoPs have been shown to be generated from the peroxidation of linolenic acid (C18:3, -3, F 1 -IsoPs), EPA (C20:5, -3, F 3 -IsoPs), and DHA (C22:6, -3, F 4 -neuroprostanes) (Fig. 3, A and B) (4,5). Similar to the distribution of F 2 -IsoP regioisomers, certain F 3 -IsoP and F 4 -neuroprostane regioisomers are generated in greater amounts than others (40).…”

Identification of Novel Autoxidation Products of the ω-3 Fatty Acid Eicosapentaenoic Acid in Vitro and in Vivo

“…In the course of performing such experiments, we also undertook studies to confirm the stability of EPA in the diet fed to mice to ensure that the source of oxidized EPA oxidation products was not the diet and that they had been generated in vivo. For these experiments, the AIN-93 diet containing EPA was quantified for F 3 -IsoPs at the beginning of the study and after its completion using the methods we have previously employed to quantify these compounds in animal tissues employing a stable isotope dilution gas chromatography MS assay (19). In addition, the diet was allowed to remain at room temperature under the same conditions as present in mouse cages for up to 7 days and F 3 -IsoPs measured.…”

“…The aqueous layer was not acidified prior to the extraction. It should be noted that we have previously shown that this extraction approach does not lead to oxidation of arachidonate or EPA ex vivo (19,25). Basic hydrolysis was carried out in 0.5 ml of MeOH and 0.5 ml of 15% KOH at 37°C for 30 min.…”

“…Analysis of F 3 -IsoPs as PFB esters was performed using the same protocol as above employing normal phase LC with a solvent system of 12% isopropyl alcohol in hexanes (32). For quantitative studies involving the measurement of F 3 -IsoPs in mouse tissues, a highly accurate gas chromatography/MS assay using stable isotope dilution methods was employed as described (19).…”