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


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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.

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

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F2 isoprostanes society for free radical biology and medicine roberts et al 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: [email protected]

F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.1


F2 isoprostanes society for free radical biology and medicine roberts et al 1

Virtual Free Radical School

Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status

Jack Roberts Josh Fessel Jason Morrow

F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.2


Free radicals in human disease

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.3


F2 isoprostanes society for free radical biology and medicine roberts et al 1

“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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.4


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.5


F2 isoprostanes society for free radical biology and medicine roberts et al 1

F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.6


Attributes of measuring f 2 isops as a biomarker of lipid peroxidation

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.7


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.8


An important concept regarding the formation and measurement of f 2 isops

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.9


Tbars assay for mda the most widely used measure of lipid peroxidation

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.10


Comparison of measurements of f 2 isops and mda

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.11


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.12


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.13


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.14


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.15


Advantages of measuring a urinary f 2 isop metabolite

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.16


F2 isoprostanes society for free radical biology and medicine roberts et al 1

-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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.17


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.18


F2 isoprostanes society for free radical biology and medicine roberts et al 1

  • 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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.19


F2 isoprostanes society for free radical biology and medicine roberts et al 1

  • 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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.20


F2 isoprostanes society for free radical biology and medicine roberts et al 1

Attack of the C• in the IsoP Pathway by O2 Leads to the Formation of “Other Products”

F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.21


Discovery of new products of lipid peroxidation

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.22


F2 isoprostanes society for free radical biology and medicine roberts et al 1

Formation of Isofuran Regioisomers

F2-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.23


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.24


F2 isoprostanes society for free radical biology and medicine roberts et al 1

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.25


Summary

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-IsoprostanesSociety For Free Radical Biology and Medicine Roberts et al.26


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