F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 1 The Isoprostane and Neuroprostane Pathways: A True View of Lipid Peroxidation Jack Roberts, Sean Davies and Jason Morrow Vanderbilt University L. Jackson Roberts, II, MD, Dept. Pharmacology, Vanderbilt University Tel: , Fax: , EM:

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 2 The Isoprostane and Neuroprostane Pathways: A True View of Lipid Peroxidation Jack Roberts Sean Davies Jason Morrow

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 3 The Isoprostane Pathway We first reported the discovery that bioactive prostaglandin F 2 -like compounds, F 2 -isoprostanes (F 2 - IsoPs), are formed non-enzymatically in vivo by free radical induced peroxidation of arachidonic acid (AA). Subsequently, we have demonstrated that several different classes of compounds are formed via the isoprostane pathway – Some exert receptor-mediated bioactivity – Some are reactive and adduct to critical biomolecules

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 4 Pathway of Formation of F 2 -IsoPs

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 5 F 2 -IsoPs are Bioactive: Effect of Intrarenal Infusion of 15-F 2t -IsoP (8-iso-PGF 2  ) on Renal Blood Flow 10 Additional F 2 -IsoPs and 15-E 2 -IsoP have been tested and all are vasoconstrictors

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 6 Intermediates in the IsoP Pathway are Prostaglandin H 2 -Like Bicyclic Endoperoxides PGH 2 is unstable, t ½ in aqueous buffer ~5 mins We have demonstrated that GSH is a key effector of the reduction of IsoP endoperoxides in vivo to F 2 - IsoPs and that other thiols can substitute However, we found that the reduction of the endoperoxides in vivo is not completely efficient – Consequently, the endoperoxides undergo rearrangement in vivo to form other products

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 7 H 2 -IsoPs Rearrange to Form E 2 -IsoPs, D 2 -IsoPs, and Isothromboxanes In Vivo

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 8 Are Cyclopentenone PGA 2 and PGJ 2 -Like IsoPs Formed In Vivo? Formed by Dehydration of E 2 - and D 2 -IsoPs Cyclopentenone prostanoids are reactive compounds They undergo Michael addition reactions with thiols, e.g. GSH, and covalently bind to proteins

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 9 Possess unique biological properties Cyclopentenone Prostanoids – Inhibit cellular proliferation due to their ability to modulate a variety of growth and stress related genes > Induce cell cycle arrest and differentiation > Induce apoptosis – Activate PPAR- 

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 10 Cyclopentenone Prostanoids Whether cyclooxygenase derived PGA 2 or PGJ 2 are actually formed in vivo has been the subject of heated controversy for over 2 decades This may be because their detection in vivo may be complicated by their sequestration as adducts, e.g. with GSH Since IsoPs are initially formed esterified on phospholipids, A 2 /J 2 -IsoPs may be shielded from this process while esterified in membranes, but would rapidly conjugate with GSH once released by phospholipase action

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 11 PLase J 2 -IsoP GSH

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 12 Time Course of Conjugation of 15-A 2t -IsoP (8-Iso- PGA 2 ) with GSH and Adduction to BSA Conjugation With GSHAdduction to BSA

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 13 Levels of A 2 /J 2 - and E 2 /D 2 -IsoPs Esterified in Rat Liver Following Administration of CCl 4 to Induce an Oxidant Injury Although E 2 /D 2 -IsoP levels increase dramatically in plasma after CCl 4, A 2 /J 2 -IsoPs could not be detected in plasma even after CCl 4, presumably because the free compounds rapidly and efficiently conjugate with thiols

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 14 Rearrangement of PGH 2 PGE 2 & PGD 2 PGH 2 Reactive molecules Adduct to proteins and form cross-links Levuglandins LGE 2 LGD 2 20% of products

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 15 Are Levuglandin-Like  -Ketoaldehydes (Isoketals) Formed v ia the IsoP Pathway?

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 16 Detection of the Formation of IsoKs Detected in abundance during oxidation of arachidonic acid in vitro However, could not be detected during oxidation of simple biological systems, e.g. LDL or microsomes – Speculated this may be due to rapid adduction to proteins – We therefore compared the rates of adduction of IsoK and 4-HNE to OVA and their ability to induce cross-links

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 17 Comparison of the Rate of Adduction to Ovalbumin and Protein Cross-Linking by IsoKs and 4-HNE OVA Control HNE IsoK (4 hrs) To detect IsoKs in biological systems, we developed a LC/MS assay for IsoK lysyl lactam adducts after enzymatic digestion of proteins to individual amino acids Adduction to OVA

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 18 Chemistry of IsoK Adduct Formation with Lysine Residues

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 19 IsoKs are Formed In Vivo : Lactam Adduct Levels in Normal Plasma Rat 199  78 pg/mL n=4 Human 561  101 pg/mL n=6 Normal levels of F 2 -IsoPs in human plasma: 35  6 pg/mL

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 20 Effect of IsoKs on Proteasome Activity and Viability of Neuroglial Cells IC50 = 330 nMLC50 = 670 nM IsoKs potenly inhibit the proteasome and are highly toxic

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 21 The Entire Isoprostane Pathway

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 22 Formation of IsoP-Like Compounds (Neuroprostanes) from Oxidation of DHA Docosahexaenoic acid (DHA) (C22:6  3) is highly enriched in brain. Oxidation of DHA forms classes of different compounds (NeuroPs) analogous to those formed by the IsoP pathway that may be sensitive biomarkers of oxidant injury in the brain and participate in mediating oxidant injury – F 4 -, E 4 -, D 4 -, A 4 -, J 4 -NeuroPs and Neuroketals are all formed and are present at readily detectable levels in normal human brain

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 23 NeuroP Regioisomers Formed From Abstraction of Specific bis- Allylic Hydrogen Atoms

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 24 Levels of F 4 -NeuroPs and NeuroKs in Brain from Patients with Alzheimer’s Disease F 4 -NeuroPs NeuroKs Both F 4 -NeuroPs and NeuroKs are significantly increased in AD brain

F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 25 Summary The IsoP and NeuroP pathways form several series of different compounds Some compounds such as F-ring and E-ring IsoPs exert potent receptor dependent biological actions Other compounds, i.e. IsoKs, NeuroKs, and A 2 /J 2 - IsoPs, are extremely reactive compounds which are cytotoxic and exert other biological effects owing to their ability to adduct to critical biomolecules