The Cyclopentenone Product of Lipid Peroxidation, 15-A _2t-Isoprostane, Is Efficiently Metabolized by HepG2 Cells via Conjugation with Glutathione

Cyclopentenone isoprostanes (IsoPs), A₂/J₂-IsoPs, are one class of IsoPs formed via the free radical-initiated peroxidation of arachidonic acid. These compounds, which are structurally similar to cyclooxygenase-derived PGA₂ and PGJ₂, contain highly reactive α,β-unsaturated carbonyl moieties. A₂/J ₂-IsoPs are generated in vivo in humans esterified in glycerophospholipids. Unlike other classes of IsoPs, however, cyclopentenone IsoPs cannot be detected in the free form; we postulated that this might be due to their rapid adduction to various thiol-containing biomolecules via Michael addition. Recently, we reported that the A-ring IsoP, 15-A_2t-IsoP, is efficiently conjugated with glutathione in vitro by certain human and rat glutathione transferases (GSTs), with the isozyme GSTA4-4 displaying the highest activity. Herein, we examined the metabolic disposition of 15-A _2t-IsoP in HepG2 cells. We report that 15-A_2t-IsoP is primarily metabolized by these cells via conjugation to glutathione. Within 6 h, approximately 60% of 15-A_2t-IsoP added to HepG2 cells was present in the form of a water soluble conjugate(s). Structural characterization of the adduct(s) by liquid chromatographytandem mass spectrometry revealed four major conjugates. These include the intact 15-A_2t-IsoP-GSH conjugate, the GSH conjugate in which the carbonyl at C-9 of 15-A_2t-IsoP is reduced, and the corresponding cysteine conjugates. These studies thus show that the primary pathway of metabolic disposition of endogenously derived cyclopentenone IsoPs occurs via conjugation with thiols.