The Quantification of Isoprostanes as an Index of Oxidant Stress In Vitro and In Vivo: Uses and Controversies Jason D.

1. The Quantification of Isoprostanes as an Index of Oxidant Stress In Vitro and In Vivo: Uses and Controversies Jason D. Morrow M.D. Vanderbilt University

2. Outline • Define what isoprostanes (IsoPs) are and how they are formed. • Methods to quantify F2-IsoPs. • Advantages and disadvantages. • Utility of measuring F2-IsoPs as indices of oxidant stress in vivo. • Effects of diseases and interventions on IsoP formation.

3. F2-Isoprostanes • Arachidonyl-containing lipids are peroxidized to PGF2-like compounds, termed F2-isoprostanes. • Formed independent of the cyclooxygenase by peroxidation of arachidonate. • Hydrolyzed from phospholipids by phospholipases including PAF acetylhydrolase. • Generated in large amounts in vivo. • Exert potent biological activity. • Effects mediated by interaction with Tx receptor.

4. 12-series 8-series 15-series 5-series Pathway of Isoprostane Formation

5. Analysis of F2-Isoprostanes • Measured either free or after liberation from tissue lipids. • Purified by Sep-Pak extraction and TLC and derivatized to PFB ester, TMS ethers. • Analyzed using stable isotope dilution techniques employing a deuteriated standard by gas chromatography/mass spectrometry. • Assay is highly robust, precise, and accurate.

6. [2H4]15-F2t-IsoP Analysis of F2-Isoprostanes in Human Plasma

7. CCl4-Mediated Oxidant Injury

8. F2-Isoprostanes Are a Reliable Marker of Lipid Peroxidation in Vivo • Comparison of formation of MDA and F2-IsoPs with hepatic injury in CCl4-treated rats.

9. F2-IsoPs as a Measure of Oxidant Stress • BOSS study (2005)-IsoPs most accurate measure of oxidant stress in CCl4-treated rats. • Deficiencies in antioxidants in vivo are associated with increased IsoP formation. • Antioxidants decrease IsoP levels in animals and humans. • IsoP levels are increased in animal models of human diseases and human disorders associated with oxidant stress.

10. Biomarkers of Oxidative Stress Study • CCl4-induced oxidant stress in rats. • Markers quantified and compared to hepatic histology: • Plasma and urine IsoPs • MDA and other measures of lipid peroxidation • Plasma antioxidants • Plasma GSH and GSSG • Protein carbonyls and specific amino acid oxidation products • 8-hydroxy-deoxyguanosine

11. Biomarkers of Oxidative Stress Study

12. Isoprostanes as an Index of Oxidant Stress in Humans-Atherosclerosis • Oxidation of lipoproteins plays a pivotal role in the development of atherosclerotic lesions. • Levels of IsoPs in oxidized LDL correlate with other markers of lipid peroxidation. • Risk factors for atherosclerosis are associated with increased levels of IsoPs in vivo in humans. • Hyperhomocysteinemia • Diabetes mellitus • Hypercholesterolemia • Chronic cigarette smoking

13. IsoP Formation is Markedly Increased In Cigarette Smokers and Abstinence Decreases Levels

14. Increases in BMI and Isoprostanes Correlate

15. Effect of Weight Loss on IsoP Formation

16. Vitamin E and Cardiovascular Disease Trials • Randomized prospective clinical trials of vitamin E in the prevention of cardiovascular disease have yielded disparate, generally negative, results. • Definitive association between oxidant stress and disease pathogenesis in humans not shown. • Studies examined subjects late in disease course. • Effect of vitamin E on oxidant stress in vivo in humans unknown. • Pharmacology of vitamin E to prevent oxidant stress has not been defined in relevant populations.

17. Time-Course Study • Administration of 3200 IU RRR--tocopherol to 8 hypercholesterolemic humans for 20 weeks. • Subjects healthy and on no lipid-lowering drugs or antioxidants. • Isoprostanes quantified at biweekly intervals.

18. Vitamin E Reduces Isoprostane Formation in Humans

19. Dose-Effect Study • Administration of 0, 100, 200, 400, 800, 1600, or 3200 IU RRR--tocopherol for 16 weeks.

20. Effect of Dose on Plasma Vitamin E Levels

21. Vitamin E Reduces Isoprostane Formation in Humans – Effect of Dose

22. Isoprostane Formation in Human Malignancy • Has not been extensively examined. • Are increased in many inflammatory conditions. • Animal models of cancer are associated with increased IsoP formation. • Rodent models of colorectal cancer. • Hepatic cancer associated with Cu/Zn SOD deficiency. • Not increased in human prostate cancer. • Cancer therapy. • Radiation therapy. • Radioiodine therapy for hyperthyroidism. • Photoimmunotherapy for T-cell lymphoma.

23. Advantages of Isoprostane Quantification to Assess Oxidant Stress • Isoprostanes are stable molecules. • The assay is highly precise and accurate. • IsoPs can be detected in all fluids and tissues. • Normal ranges can be defined. • Allows for studies to evaluate the effects of interventions on endogenous lipid peroxidation.

24. Technical Issues Related to Isoprostane Quantification Using Mass Spectrometry • Precision - + 6%. • Accuracy – 96% • Interday and intraday variability <12%. • Diurnal variation – none at the group level in large studies; does exist within individuals. • Daily variation - <15% • Assay has been standardized across labs.

25. Disadvantages of Isoprostane Quantification to Assess Oxidant Stress • Samples must either be analyzed immediately or stored at –70o C. • Increases in IsoPs locally in tissues or fluids aren’t detected by measuring systemic oxidant stress.

26. Isoprostanes Are Increased Selectively in the Central Nervous System of Humans with AD

27. Disadvantages of Isoprostane Quantification to Assess Oxidant Stress • Samples must either be analyzed immediately or stored at –70o C. • Increases in IsoPs locally in tissues or fluids aren’t detected by measuring systemic oxidant stress. • F2-IsoPs represents only one of a myriad of arachidonate oxygenation products.

28. Classes of IsoPs

29. Disadvantages of Isoprostane Quantification to Assess Oxidant Stress • Samples must either be analyzed immediately or stored at –70o C. • Increases in IsoPs locally in tissues or fluids aren’t detected by measuring systemic oxidant stress. • F2-IsoPs represents only one of a myriad of arachidonate oxygenation products. • Analysis is labor intensive and requires expensive equipment.

30. Immunoassay Methods to Quantify IsoPs • Immunoassays advantageous because they are more economical and less labor intensive. • Polyclonal antibodies have been made by several investigators and are commercially available. • Accurate quantification using immunoassays requires initial compound purification. • Amounts measured by immunoassays often differ from those obtained by mass spectrometry. • We are currently collaborating with Unilever Ltd. in the generation of highly specific monoclonal abs.

31. Disadvantages of Isoprostane Quantification to Assess Oxidant Stress • Samples must either be analyzed immediately or stored at –70o C. • Increases in IsoPs locally in tissues or fluids aren’t detected by measuring systemic oxidant stress. • F2-IsoPs represents only one of a myriad of arachidonate oxygenation products. • Analysis is labor intensive and requires expensive equipment. • Urinary IsoPs are not a valid measure of systemic oxidant stress since a major source of urinary IsoPs is likely the kidney.

32. Metabolic Fate of 15-F2t-IsoP (8-iso-PGF2a) in Humans

33. Summary • Quantification of F2-isoprostanes is an accurate measure of lipid peroxidation in vitro and in vivo. • Measurement has provided insights into role of oxidant stress in disease and that antioxidants and other interventions can decrease endogenous lipid oxidation. • Current methods employ mass spectrometry and immunoassays. • The development of an assay to measure F2-IsoP-M may offer certain advantages over the quantification of parent F2-IsoPs.