# Presentation: 179428

## 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

- Jason D. Morrow M.D.
-  Vanderbilt University

## 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.

## 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.

## Pathway of Isoprostane Formation

## 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.

## Analysis of F2-Isoprostanes in Human Plasma  

## CCl4-Mediated Oxidant Injury

## 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.

## 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.

## 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

## Biomarkers of Oxidative Stress Study

## 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

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

## Increases in BMI and Isoprostanes Correlate

## Effect of Weight Loss on IsoP Formation

## 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.

## 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.

## Vitamin E Reduces Isoprostane Formation in Humans 

## Dose-Effect Study

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

## Effect of Dose on Plasma Vitamin E Levels

## Vitamin E Reduces Isoprostane Formation in Humans – Effect of Dose

## 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.

## 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.

## 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.

## 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.

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

## 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.

## Classes of IsoPs

## 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.

## 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.

## 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.

## Metabolic Fate of 15-F2t-IsoP (8-iso-PGF2) in Humans

## 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.