How the Serendipitous Discovery of Isoprostanes Advanced the Field of Free Radical Biology and Medic

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How the Serendipitous Discovery of Isoprostanes
Advanced the Field of Free Radical Biology and
Medicine
L. Jackson Roberts, II, MD
Jason D. Morrow, MD
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Outline

• Discovery of F2-IsoPs and biochemical pathways

• F2-IsoPs as biomarkers of oxidative stress/injury

• Bioactivities of products of the IsoP pathway

• IsoP-like compounds from other fatty acids

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Yearly Publications Involving Isoprostanes
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How Did We Discover Isoprostanes?
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PGD2 Isomerization and11-Ketoreductase Metabolism
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Analysis of Plasma for PGF2 Isomer Metabolites of
PGD2
B
100
A
50
100
2H7 9?,11?-PGF2
2H7 9?,11?-PGF2
50
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Analysis of Plasma for PGF2 Isomer Metabolites of
PGD2
m/z 569 2000 pg/ml
B
100
A
50
100
2H7 9?,11?-PGF2
2H7 9?,11?-PGF2
50
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So We Looked Around for More Normal Plasma That
Had Been Stored at -20O C in Our Colleagues
Freezers and They Kept Asking Us What Are You
Doing?
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Non-Enzymatic Mechanism of Formation of
Prostanoids
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Unique Aspect of the Formation of IsoPs

• We recently showed that PAF-AH is the enzyme
  responsible for hydrolyzing IsoPs from
  phospholipids (JBC 2814616, 2006)

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Space Filling Model of an F2-IsoP-Containing
Phospholipid
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Formation of E-Ring and D-Ring IsoPs
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Formation of A-Ring and J-Ring IsoPs
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Formation of Isothromboxanes
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Formation of E2- and D2-Isoketals
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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.

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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.

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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.
• Chronic cigarette smoking
• Hyperhomocysteinemia
• Diabetes mellitus
• Obesity

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IsoP Formation in Humans Increased IsoP Levels
in Cigarette Smokers and Effect of Abstinence
Non-Smokers vs. Smokers
Effect of Abstinence
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Increases in BMI and Isoprostanes Correlate
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Effect of Weight Loss on IsoP Formation
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Effect of Acute Caloric Restriction on IsoP
Formation
Roberts et al. (unpublished)
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Neurodegeneration and Oxidant Stress

• Oxidant stress has been implicated in
  neurodegenerative diseases.
• Amyotrophic lateral sclerosis
• Huntingtons Disease
• Alzheimers Disease
• Increased levels of lipid peroxidation products
  in brains of AD patients post mortem.
• 4-hydroxynonenal
• Malondialdehyde
• 4-hydroxy-2-hexenal
• Prior to the late 1990s, no studies had
  accurately assessed markers of CNS oxidation
  intra vitam.

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F2-Isoprostane Levels in CSF From Living Patients
with AD
Neurology 52562,1999
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Longitudinal Study of CSF IsoP Levels in AD
Patients

• Rationale
• Previous studies show substantial overlap between
  IsoP levels in AD and control patients.
• Other CNS inflammatory biomarkers increase or
  decrease as AD progresses.
• Study Design
• Prospective.
• N40 patients with very early AD.
• Patients received either vitamin E, vitamin E C
  or neither.
• CSF fluid sampled at 0, 4 and 12 months of
  diagnosis.

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CSF IsoPs Increase with AD Progression
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Antioxidants Affect CSF IsoP Levels in Humans
with AD
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F2-IsoPs Are Not Merely Markers of Oxidant Injury
- They Are Also Bioactive Compounds

• A distinguishing feature between F2-IsoPs and PGs
  is that the vast majority of the side chains of
  IsoPs are oriented cis whereas in PGs they are
  always trans

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Synthetic F2-IsoPs Tested for Activity in Rat
Retinal and Cerebral Vasculature
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EC50s for Contraction of Retinal Vasculature by
F2-IsoPs
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Receptor-Mediated Effects of 15-F2t-IsoP

• In the vasculature, the effects 15-F2t-IsoP are
  consistently abrogated by thromboxane receptor
  antagonists

• However, in the platelet, it acts as an
  antagonist of thromboxane receptor agonists

• So - some think there is a distinct IsoP
  receptor, but others arent so sure

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Effects of F2-IsoPs on Cerebral Endothelial Cells

• In the cerebral vascular the F2-IsoPs are not
  only potent vasoconstrictors, they cause cerebral
  endothelial cell death involving a positive
  feedback loop with TxA2

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Reactive Compounds Formed Via the Isoprostane
Pathway

• Cyclopentenone isoprostanes

• Undergo Michael addition with thiols

• Inhibit the inflammatory response in macrophages

• Enhance neurodegeneration

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Proposed Mechanism of CP-IsoP Induced Neuronal
Death
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Reactive Compounds Formed Via the Isoprostane
Pathway

• Isoketals

• Form covalent adducts with lysine residues

• Crosslink proteins

• Inhibit proteasome function

• Cytotoxic

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IsoK Lysyl-Adduct Pathway
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Comparison of the Rate of Adduction to Protein
and Protein Cross-Linking of IsoKs and 4-HNE
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Time-Course of IsoK-Induced Cross-Linking of OVA
IsoK-Induced Cross-Linking of Protein
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IHC of Alzheimers Disease Brain with a Single
Chain Antibody that Recognizes IsoK Lysyl Adducts
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Neuroprostanes A Specific Marker of Neuronal
Injury Derived from Docosahexaenoic Acid
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Neuroprostane Formation is a More Sensitive
Indicator of Oxidant Stress than Isoprostanes in
Humans with AD
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Formation of F3-IsoPs In Vitro and In Vivo
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Hypothesis

• Supplementation of animals and humans with fish
  oil (EPA) results in
• Decreased production of pro-inflammatory
  arachidonate-derived 2-series IsoPs.
• Increased formation of anti-inflammatory 3-series
  IsoPs from EPA.

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Effect of EPA Supplementation on 2-Series and
3-Series IsoP Formation in Mice
F2-IsoPs
F3-IsoPs
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Effect of Fish Oil Supplementation on 2-Series
IsoP Formation in Humans
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Cyclopentenone PGs Activate Nrf2 Signaling and
Antioxidant Enzymes
15-deoxy-?-12,14-PGJ2
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Formation of Cyclopentenone Isoprostanes
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Formation of Cyclopentenone IsoP Adducts with GSH
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Methods

• Determine whether oxidized EPA contains products
  that can activate Nrf2.
• EPA oxidized in vitro.
• Translocation of Nrf2 to nucleus via
  immunoblotting in HepG2 cells.
• Assess Nrf2 translocation via a flow cytometric
  assay utilizing HepG2 cells containing an ARE-GFP
  reporter.

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Oxidized EPA Induces ARE-GFP Reporter Activity
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Oxidized EPA Stabilizes Nrf2
oxidized native
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HPLC Separation of Bioactive Oxidized EPA
Fractions

ETOH
24
22
26
20
21
25
27
28
29
23
()
Nrf2
Actin
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HPLC Separation of Bioactive Oxidized EPA
Fractions (25)
RT
5.00 - 9.00
SM
7G
100
95
90
m/z 331
85
80
75
70
65
60
55
50
45
40
35
Relative Abundance
30
25
20
15
10
5
0
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
Time (min)
-H2O
-H2O-CO2
A3/J3-IsoPs
313
100
95
269
90
85
80
75
70
65
60
55
50
15-series
45
M-H-
40
35
331
215
Relative Abundance
30
25
20
-2H2O
189
15
295
10
5
83.0
371.7
59.1
0
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J-Ring IsoPs Generated from EPA Peroxidation Are
Potent Inducers of ARE-Driven GFP Expression
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Conclusions

• The isoprostanes are a novel series of
  arachidonate peroxidation products that possess
  potent bioactivity and may mediate some of the
  adverse effects associated with oxidant stress.
• 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.
• Isoprostane-like molecules can be generated from
  other polyunsaturated fatty acids and these
  compounds may play a role in the pathophysiology
  of oxidant stress.