Discovery of lipid peroxidation products formed in vivo with a substituted tetrahydrofuran ring (isofurans) that are favored by increased oxygen tension

Abstract

Free radicals have been implicated in the pathogenesis of an increasing number of diseases. Lipids, which undergo peroxidation, are major targets of free radical attack. We report the discovery of a pathway of lipid peroxidation that forms a series of isomers in vivo that are characterized by a substituted tetrahydrofuran ring structure, termed isofurans (IsoFs). We have proposed two distinct pathways by which IsoFs can be formed based on 18O2 and H2 18O labeling studies. Measurement of F2-isoprostanes (IsoPs), prostaglandin F2-like compounds formed nonenzymatically as products of lipid peroxidation, is considered one of the most reliable approaches for assessing oxidative stress status in vivo. However, one limitation with this approach is that the formation of IsoPs becomes limited at high oxygen tension. In contrast, the formation of IsoFs becomes increasingly favored as oxygen tension increases. IsoFs are present at readily detectable levels in normal fluids and tissues, and levels increase dramatically in CCl4-treated rats, an animal model of oxidant injury. The ratio of IsoFs to IsoPs in major organs varies according to normal steady-state tissue oxygenation. In addition, IsoFs show a marked increase early in the course of hyperoxia-induced lung injury, whereas IsoPs do not significantly increase. We propose that combined measurement of IsoFs and IsoPs should provide a more reliable index of oxidant stress severity than quantification of either alone because of the opposing modulation of the two pathways by oxygen tension, which can vary widely in different organs and disease states.

Fig 1.

Pathway leading to the formation of IsoPs and other arachidonic acid-derived products of lipid peroxidation. Attack of the carbon-centered radical intermediate by oxygen prevents the formation of IsoPs while favoring the formation of other products.

Fig 2.

Electron ionization mass spectrum of IsoFs isolated from an incubation after oxidation of arachidonic acid in vitro. Compounds were analyzed as a methyl ester, TMS ether derivative. In this mixed mass spectrum, the IsoF regioisomer shown appears to predominate this part of the spectrum, by way of assignment of the m/z 181 base ion. OTMS and TMSO indicate trimethylsilyl ether groups. The assignment of fragment ions to other regioisomers is outlined in Fig. 8.

Fig 3.

Effect of oxygen tension on F₂-IsoP and IsoF formation during oxidation of arachidonic acid in vitro. Each bar represents the mean ± SEM for three independent experiments. *, P < 0.05; **, P < 0.001.

Fig 4.

IsoF and F₂-IsoP levels in normal rat organs and liver from rats treated with CCl₄. (A) Levels of IsoFs and F₂-IsoPs esterified in liver 4 h after administration of CCl₄ and in untreated animals. Results are the mean ± SEM for three animals each in the untreated and treated groups. (B) Ratios of IsoF/F₂-IsoP levels measured in rat brain (n = 4), kidney (n = 6), and liver (n = 3). Brain represents rat brain hippocampus, one of the most highly oxygenated regions of the brain. Results are shown as the mean ± SEM. (C) Effect of antioxidants on IsoF formation. Results are shown as the mean ± SEM. Control are normal rats (n = 8), NAC are rats treated with N-acetylcysteine (n = 4), and LA are rats treated with α-lipoic acid (n = 6). *, P < 0.05 vs. control.

Fig 5.

IsoF and F₂-IsoP levels esterified in lung tissue at baseline and after 3 h of hyperoxia. Results are shown as mean ± SEM. *, P < 0.001 vs. IsoF baseline; #, P < 0.01 vs. IsoP 3 h, n = 8 in each group.