Mechanisms for the Formation of Isoprostane Endoperoxides from Arachidonic Acid

Abstract

The isoprostanes are a class of autoxidation products generated from arachidonic acid (or its esters) by a free radical initiated process. The potent biological activity of these compounds has been attracting intense research interest since they were detected in humans as well as animal models in the early 1990s. The measurement of these compounds has been regarded as one of the most useful non-invasive biomarkers for oxidative stress status. Two mechanisms for the formation of these compounds have been proposed. In the first mechanism, a peroxyl radical undergoes successive 5-exo cyclizations analogous to the enzymatic mechanism proposed for prostaglandin biosynthesis. The second mechanism starts with a 4-exo cyclization of a peroxyl radical leading to an intermediate dioxetane, a mechanism that has also been proposed for prostaglandin biosynthesis as well as for the formation of 4-hydroxy nonenal (HNE). Autoxidation of cholesteryl-15-HpETE under free radical conditions provides Type IV isoprostanes. The "dioxetane" mechanism for isoprostane generation from 15-HpETE requires that optically pure products are formed from an optically pure reactant, whereas an alternate mechanism for the process involving ␤-fragmentation of the 15-peroxyl would give racemic isoprostane products. We have carried out a test of the mechanism based upon these stereochemical requirements. The results of analysis of the product mixture derived from autoxidation of optically pure Ch-15-HpETE by atmospheric pressure chemical ionizationmass spectrometry coupled with chiral high performance liquid chromatography indicate that the major isoprostane diastereomers are formed as a racemic mixture. These experimental results are consistent with a mechanism for isoprostane formation involving ␤-fragmentation of the 15-peroxyl radical followed by re-addition of oxygen to form the 11-HPETE peroxyl, and they exclude a mechanism proceeding through the formation of a dioxetane intermediate.

Identification of a Novel Class of Endoperoxides from Arachidonate Autoxidation

“…Consequently, the set of products derived only from the 12-peroxyl radical form when 12-HPETE is used as a starting material. Characterization of the oxidation products that derive from the individual hydroperoxides greatly simplifies the analytical problem and provides mechanistic insights for the autoxidation process (13).…”

“…The PFB ester was separated by flash chromatography using ethyl acetate and hexane (1:4). The structure of the product was characterized by 1 H, 13 C, and 19 F NMR. The 1,3-diol on the cyclopentane ring was protected by formation of butylboronate.…”

“…Formation of regioisomeric and diastereoisomeric isoprostanes has been extensively studied (12,13). Previous experimental observations showed that 5-and 15-series (numbers refer to the position of hydroxyl group on the side chain) isoprostanes are more abundant than those of 8-and 12-series (12, 19 -21).…”

“…A number of oxidation products from arachidonate autoxidation have been characterized and their biological activities are a major research focus (Scheme 1). Bicyclic endoperoxides (1a), monocyclic peroxides (1b), and serial cyclic peroxides (1c) have previously been characterized from the mixture of products formed in the autoxidation of cholesteryl arachidonate (8,12,13). Oxidation products having a tetrahydrofuran moiety (1d) were recently characterized under conditions of reaction having increased oxygen tension (14).…”

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

“…Free radical-induced oxidation of arachidonic acid has been well studied and a unified mechanism that leads to the formation of various oxidation products has been proposed (Scheme 2) (21)(22)(23). The oxidation proceeds via hydrogen atom abstraction at one of three bis-allylic positions at carbon 7, 10, and 13 to form a pentadienyl radical that can be trapped by molecular oxygen to form a peroxyl radical.…”