The Isoprostane and Neuroprostane Pathways: A True View of Lipid Peroxidation

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: 615-343-1816, Fax: 615-343-9446, EM: jack.roberts@vanderbilt.edu F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.1

2. The Isoprostane and Neuroprostane Pathways: A True View of Lipid Peroxidation Jack Roberts Sean Davies Jason Morrow F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.2

3. The Isoprostane Pathway • We first reported the discovery that bioactive prostaglandin F2-like compounds, F2-isoprostanes (F2-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 F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.3

4. Pathway of Formation of F2-IsoPs F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.4

5. F2-IsoPs are Bioactive: Effect of Intrarenal Infusion of 15-F2t-IsoP (8-iso-PGF2) on Renal Blood Flow 10 Additional F2-IsoPs and 15-E2-IsoP have been tested and all are vasoconstrictors F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.5

6. Intermediates in the IsoP Pathway are Prostaglandin H2-Like Bicyclic Endoperoxides • PGH2 is unstable, t½ in aqueous buffer ~5 mins • We have demonstrated that GSH is a key effector of the reduction of IsoP endoperoxides in vivo to F2-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 F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.6

7. H2-IsoPs Rearrange to Form E2-IsoPs, D2-IsoPs, and Isothromboxanes In Vivo F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.7

8. Cyclopentenone prostanoids are reactive compounds • They undergo Michael addition reactions with thiols, e.g. GSH, and covalently bind to proteins Are Cyclopentenone PGA2 and PGJ2-Like IsoPs Formed In Vivo? Formed by Dehydration of E2- and D2-IsoPs F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.8

9. Cyclopentenone Prostanoids • Possess unique biological properties • 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- F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.9

10. Cyclopentenone Prostanoids • Whether cyclooxygenase derived PGA2 or PGJ2 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. withGSH • Since IsoPs are initially formed esterified on phospholipids, A2/J2-IsoPs may be shielded from this process while esterified in membranes, but would rapidly conjugate with GSH once released by phospholipase action F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.10

11. J2-IsoP PLase GSH GSH GSH GSH GSH GSH F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.11

12. Conjugation With GSH Adduction to BSA Time Course of Conjugation of 15-A2t-IsoP (8-Iso-PGA2) with GSH and Adduction to BSA F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.12

13. Levels of A2/J2- and E2/D2-IsoPs Esterified in Rat Liver Following Administration of CCl4 to Induce an Oxidant Injury Although E2/D2-IsoP levels increase dramatically in plasma after CCl4, A2/J2-IsoPs could not be detected in plasma even after CCl4, presumably because the free compounds rapidly and efficiently conjugate with thiols F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.13

14. PGH2 PGE2 & PGD2 LGE2 • Reactive molecules • Adduct to proteins and form cross-links 20% of products LGD2 Levuglandins Rearrangement of PGH2 F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.14

15. Are Levuglandin-Like -Ketoaldehydes (Isoketals) Formed via the IsoP Pathway? F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.15

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 F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.16

17. Adduction to OVA (4 hrs) HNE Control IsoK OVA Comparison of the Rate of Adduction to Ovalbumin and Protein Cross-Linking by IsoKs and 4-HNE 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 F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.17

18. Chemistry of IsoK Adduct Formation with Lysine Residues F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.18

19. Rat Human 199  78 pg/mL 561  101 pg/mL n=4 n=6 IsoKs are Formed In Vivo: Lactam Adduct Levels in Normal Plasma Normal levels of F2-IsoPs in human plasma: 35  6 pg/mL F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.19

20. Effect of IsoKs on Proteasome Activity and Viability of Neuroglial Cells IC50 = 330 nM LC50 = 670 nM IsoKs potenly inhibit the proteasome and are highly toxic F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.20

21. The Entire Isoprostane Pathway F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.21

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 • F4-, E4-, D4-, A4-, J4-NeuroPs and Neuroketals are all formed and are present at readily detectable levels in normal human brain F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.22

23. NeuroP Regioisomers Formed From Abstraction of Specific bis-Allylic Hydrogen Atoms F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.23

24. F4-NeuroPs NeuroKs Levels of F4-NeuroPs and NeuroKs in Brain from Patients with Alzheimer’s Disease Both F4-NeuroPs and NeuroKs are significantly increased in AD brain F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.24

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 A2/J2-IsoPs, are extremely reactive compounds which are cytotoxic and exert other biological effects owing to their ability to adduct to critical biomolecules F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.25