Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status

1. Virtual Free Radical School Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts , Josh Fessel, and Jason Morrow Vanderbilt University 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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.1

2. Virtual Free Radical School Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts Josh Fessel Jason Morrow F2-IsoprostanesOxygen Society EducationProgram Roberts et al.2

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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.3

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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.4

5. F2-Isoprostanes Discovered One Year Later They are: • PGF2-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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.5

6. F2-IsoprostanesOxygen Society EducationProgram Roberts et al.6

7. Attributes of Measuring F2-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% F2-IsoprostanesOxygen Society EducationProgram Roberts et al.7

8. Attributes of Measuring F2-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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.8

9. An Important Concept Regarding the Formation and Measurement of F2-IsoPs • Initially formed in situ on phospholipids (PL) and then released by phospholipase action • Levels of free F2-IsoPs, e.g. in plasma, provides an index of total endogenous production of IsoPs • Levels of esterified F2-IsoPs can localize oxidant injury in key tissues/organs of interest F2-IsoprostanesOxygen Society EducationProgram Roberts et al.9

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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.10

11. Comparison of Measurements of F2-IsoPs and MDA • In vitro during oxidation of rat liver microsomes • In vivo in rat liver following administration of CCl4 to induce a severe oxidant injury to the liver: F2-IsoprostanesOxygen Society EducationProgram Roberts et al.11

12. In Vitro In Vivo The formation of both in vitro correlate well - note there is ~25,000 times more MDA than F2-IsoPs However, in vivo levels of F2-IsoPs detected are ~28-fold higher than MDA Comparison of Measurements of F2-IsoPs and MDA F2-IsoprostanesOxygen Society EducationProgram Roberts et al.12

13. An Example of the Value of Measuring F2-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) F2-IsoprostanesOxygen Society EducationProgram Roberts et al.13

14. Roberts LJ, II & Reckelhoff JA BBRC 287:254, 2001 Plasma Concentrations of F2-IsoPs in Young and Aged Rats Measurement of F2-IsoPs unveils profound oxidant stress in aged rats F2-IsoprostanesOxygen Society EducationProgram Roberts et al.14

15. Limitations of Measuring Unmetabolized F2-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 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.15

16. Advantages of Measuring a Urinary F2-IsoP Metabolite • Can provide a time-integrated index of total endogenous IsoP production by collecting urine over several hours • Important in situations where there are large fluctuations in the extent of oxidant injury over time • Measured in urine • Eliminates need for blood collection • Metabolites cannot be formed ex vivo by autoxidation • No special storage conditions are required F2-IsoprostanesOxygen Society EducationProgram Roberts et al.16

17. -oxidation 5-reduction Major urinary metabolite (Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996) Metabolic Fate of 15-F2t-IsoP (8-Iso-PGF2) in Humans A GC/MS assay for F2-IsoP-M has been developed F2-IsoprostanesOxygen Society EducationProgram Roberts et al.17

18. Plasma Esterified Plasma Free Urinary F2-IsoP-M Assessments of F2-IsoP Production in Patients With Polygenic Hypercholesterolemia The relative increases in F2-IsoPs assessed by all 3 approaches are in close agreement F2-IsoprostanesOxygen Society EducationProgram Roberts et al.18

19. 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 • O2 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 Partial List of Disorders in Which Measurements Of F2-IsoPs Has Implicated a Pathogenic Role for Oxidative Stress F2-IsoprostanesOxygen Society EducationProgram Roberts et al.19

20. Competing with this is attack of the [C] by molecular O2 [C+ O2 COO]to form other products Potential Limitation of Measuring F2-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 • Accordingly, measurement of F2-IsoPs is an insensitive marker of lipid peroxidation in setting of high O2 tension, e.g. hyperoxic lung injury F2-IsoprostanesOxygen Society EducationProgram Roberts et al.20

21. • • • • • • Attack of the C• in the IsoP Pathway by O2 Leads to the Formation of “Other Products” F2-IsoprostanesOxygen Society EducationProgram Roberts et al.21

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 F2-IsoPs becomes highly disfavored at elevated oxygen tensions, the formation of IsoFs becomes highly favored F2-IsoprostanesOxygen Society EducationProgram Roberts et al.22

23. • • Formation of Isofuran Regioisomers F2-IsoprostanesOxygen Society EducationProgram Roberts et al.23

24. Effect of O2 Tension on the Formation of F2-IsoPs and IsoFs During Oxidation of Arachidonic Acid In Vitro The formation of IsoFs becomes highly favored at high concentrations of O2 F2-IsoprostanesOxygen Society EducationProgram Roberts et al.24

25. The Ratio of IsoFs to F2-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) F2-IsoprostanesOxygen Society EducationProgram Roberts et al.25

26. Summary • The formation of F2-IsoPs and IsoFs are differentially modulated by oxygen tension • Accordingly, combined measurement of both F2-IsoPs and IsoFs may provide the best assessment of lipid peroxidation and oxidative stress status F2-IsoprostanesOxygen Society EducationProgram Roberts et al.26