1 / 26

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

Virtual Free Radical School. Measurement of F 2 -Isoprostanes and Isofurans as Indices of Oxidative Stress Status. Jack Roberts , Josh Fessel, and Jason Morrow Vanderbilt University.

anneke
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related