1. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 1 Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts, Josh Fessel, and Jason Morrow Vanderbilt University Virtual Free Radical School L. Jackson Roberts, II, MD, Dept. Pharmacology, Vanderbilt University, Nashville, TN 37232 Tel: 615-343-1816, Fax: 615-343-9446, EM: jack.roberts@vanderbilt.edu

2. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 2 Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts Josh Fessel Jason Morrow Virtual Free Radical School

3. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 3 Free Radicals in Human Disease Thought to play a causative role in a wide variety of human diseases ranging from cancer, neurodegenerative disease, atherosclerosis, and even the normal aging process However, a major impediment in translating these hypotheses to fact has been the lack of a reliable approach to assess oxidative stress status in humans – This is because most methods developed a lack in specificity and/or sensitivity or are too invasive for human investigation

4. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 4 “One of the greatest needs in the field now is the availability of a non-invasive test to probe the oxidative stress status of humans” William A. Pryor, Co-Editor-In Chief Free Radical Biology & Medicine, 1989

5. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 5 F 2 -Isoprostanes Discovered One Year Later PGF 2 -like compounds produced non-enzymatically by free radical-induced peroxidation of arachidonic acid (AA) Produced in abundance in vivo in quantities far exceeding cyclooxygenase-derived PGs Capable of exerting potent biological actions 4 regioiosmers are formed (5-, 8-, 12-, and 15-series), each of which are comprised of 8 racemic diastereomers They are:

6. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 6

7. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 7 Attributes of Measuring F 2 -IsoPs as a Biomarker of Lipid Peroxidation Specific products of lipid peroxidation Very stable Detectable in all normal biological fluids and tissues – Thus allowing the definition of a normal range NICI GC/MS assay has a lower limit of detection in the low pg range, a precision of  6%, and an accuracy of 96%

8. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 8 Attributes of Measuring F 2 -IsoPs as a Biomarker of Lipid Peroxidation Their formation increases dramatically in animal models of oxidant injury Their formation is modulated by antioxidant status – Impairment of endogenous antioxidants enhances their formation – Administration of antioxidant agents suppresses their formation

9. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 9 An Important Concept Regarding the Formation and Measurement of F 2 -IsoPs Initially formed in situ on phospholipids (PL) and then released by phospholipase action Levels of free F 2 -IsoPs, e.g. in plasma, provides an index of total endogenous production of IsoPs Levels of esterified F 2 -IsoPs can localize oxidant injury in key tissues/organs of interest

10. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 10 TBARS Assay for MDA: The Most Widely Used Measure of Lipid Peroxidation Popular – Because it can even be performed by the intellectually impaired Problems – The TBARS assay is not specific for MDA – MDA is not a specific product of lipid peroxidation

11. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 11 Comparison of Measurements of F 2 -IsoPs and MDA In vitro d uring oxidation of rat liver microsomes In vivo in rat liver following administration of CCl 4 to induce a severe oxidant injury to the liver:

12. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 12 Comparison of Measurements of F 2 -IsoPs and MDA In Vitro The formation of both in vitro correlate well - note there is ~25,000 times more MDA than F 2 -IsoPs In Vivo However, in vivo levels of F 2 -IsoPs detected are ~28-fold higher than MDA

13. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 13 An Example of the Value of Measuring F 2 -IsoPs The free radical theory of aging is a very popular theory However, indices of oxidative stress measured in young and old animals, e.g. MDA and protein carbonyls, have in most cases yielded conflicting or unimpressive differences (only 2- to 3-fold or less)

14. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 14 Roberts LJ, II & Reckelhoff JA BBRC 287:254, 2001 Plasma Concentrations of F 2 -IsoPs in Young and Aged Rats Measurement of F 2 -IsoPs unveils profound oxidant stress in aged rats

15. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 15 Limitations of Measuring Unmetabolized F 2 -IsoPs Does not provide a time-integrated index of IsoP production Precautions must be taken to prevent formation of IsoPs ex vivo by autoxidation in some biological fluids, e.g. plasma, during storage Unmetabolized IsoPs in urine can, in part, reflect renal rather than systemic production of IsoPs

16. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 16 Advantages of Measuring a Urinary F 2 -IsoP Metabolite Can provide a time-integrated index of total endogenous IsoP production by collecting urine over several hours Metabolites cannot be formed ex vivo by autoxidation – No special storage conditions are required Measured in urine – Eliminates need for blood collection – Important in situations where there are large fluctuations in the extent of oxidant injury over time

17. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 17 Metabolic Fate of 15-F 2t -IsoP (8-Iso-PGF 2  ) in Humans A GC/MS assay for F 2 -IsoP-M has been developed (Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996)  -oxidation  5 -reduction Major urinary metabolite

18. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 18 Plasma Esterified Plasma Free Assessments of F 2 -IsoP Production in Patients With Polygenic Hypercholesterolemia Urinary F 2 -IsoP-M The relative increases in F 2 -IsoPs assessed by all 3 approaches are in close agreement

19. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 19 Partial List of Disorders in Which Measurements Of F 2 -IsoPs Has Implicated a Pathogenic Role for Oxidative Stress Smoking Alzheimer’s disease Hunington’s disease Atherosclerosis Hepatorenal syndrome Scleroderma Cardiac/renal ischemia/reperfusion injury Coronary angioplasty Se and vitamin E deficiency Organophosphate poisoning Hyperhomocysteinemia Renal transplant dysfunction Diabetes Rhabdomyolysis Bile duct obstruction O 2 pulmonary toxicity in premature infants Halothane hepatotoxicity Acetaminophen poisoning Age-related decline in renal function Cr (VI) poisoning Retinopathy of the newborn Alcohol ingestion Paraquat poisoning Cisplatin-induced renal dysfunction

20. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 20 Potential Limitation of Measuring F 2 -IsoPs As An Index of Oxidative Stress An intermediate in the formation of IsoPs is a carbon-centered radical [C  ] Intramolecular attack by this radical is required to form the cyclopentane ring in IsoPs Competing with this is attack of the [C  ] by molecular O 2 [C  + O 2 COO  ] to form other products Accordingly, measurement of F 2 -IsoPs is an insensitive marker of lipid peroxidation in setting of high O 2 tension, e.g. hyperoxic lung injury

21. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 21 Attack of the C in the IsoP Pathway by O 2 Leads to the Formation of “Other Products”

22. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 22 Discovery of New Products of Lipid Peroxidation We recently discovered the formation of a series of compounds with a substituted tetrahydrofuran ring (Isofurans) (Fessel JP & Roberts LJ, II, et. al. submitted) which are products of oxygen attack on the carbon centered radical in the IsoP pathway. – Whereas the formation of F 2 -IsoPs becomes highly disfavored at elevated oxygen tensions, the formation of IsoFs becomes highly favored

23. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 23 Formation of Isofuran Regioisomers

24. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 24 Effect of O 2 Tension on the Formation of F 2 -IsoPs and IsoFs During Oxidation of Arachidonic Acid In Vitro The formation of IsoFs becomes highly favored at high concentrations of O 2

25. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 25 The Ratio of IsoFs to F 2 -IsoPs Correlates with Tissue Oxygenation In Vivo Levels of IsoFs are higher than IsoPs in highly oxygenated organs (brain and kidney) but lower in poorly oxygenated organs (liver)

26. F 2 -Isoprostanes Society For Free Radical Biology and Medicine Roberts et al. 26 Summary Accordingly, combined measurement of both F 2 -IsoPs and IsoFs may provide the best assessment of lipid peroxidation and oxidative stress status The formation of F 2 -IsoPs and IsoFs are differentially modulated by oxygen tension