Transcript The Isoprostane and Neuroprostane Pathways: A True View of Lipid Peroxidation Jack Roberts, Sean Davies and Jason Morrow Vanderbilt University L.

The Isoprostane and Neuroprostane Pathways:
A True View of Lipid Peroxidation
Jack Roberts, Sean Davies and Jason Morrow
Vanderbilt University
L. Jackson Roberts, II, MD, Dept. Pharmacology, Vanderbilt University
Tel: 615-343-1816, Fax: 615-343-9446, EM: [email protected]
F2-Isoprostanes
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Roberts et al. 1
The Isoprostane and Neuroprostane Pathways:
A True View of Lipid Peroxidation
Jack Roberts
F2-Isoprostanes
Sean Davies
Jason Morrow
Society For Free Radical Biology and Medicine
Roberts et al. 2
The Isoprostane Pathway
•
We first reported the discovery that bioactive
prostaglandin F2-like compounds, F2-isoprostanes (F2IsoPs), are formed non-enzymatically in vivo by free
radical induced peroxidation of arachidonic acid (AA).
•
Subsequently, we have demonstrated that several
different classes of compounds are formed via the
isoprostane pathway
– Some exert receptor-mediated bioactivity
– Some are reactive and adduct to critical biomolecules
F2-Isoprostanes
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Roberts et al. 3
Pathway of Formation of F2-IsoPs
AA
COOH
.
.
COOH
.
OO
OO
..
COOH
O2
O2
COOH
O2
O2
.
COOH
COOH
COOH
COOH
.
O O
.
O O
.
O
COOH
O
.
O
O
COOH
.
O
COOH
O2
O2
O2
OOH
OOH
COOH
O
O
COOH
O
O
COOH
O
COOH
.
O
O
O2
O
O
COOH
O
O
OOH
OOH
[H]
OH
OH
OH
5
OH
COOH
COOH
5-Series
OH
12-Series
OH
OH
8
12
OH
OH
F2-Isoprostanes
[H]
[H]
[H]
COOH
COOH
15
OH
OH
8-Series
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OH
15-Series
Roberts et al. 4
F2-IsoPs are Bioactive: Effect of Intrarenal Infusion of
15-F2t-IsoP (8-iso-PGF2) on Renal Blood Flow
Decrease in Renal Blood Flow (%)
0
20
40
60
80
100
0.0
0.5
1.0
1.5
2.0
Dose of 15-F2t-IsoP Infused
10 Additional F2-IsoPs and 15-E2-IsoP have been tested and all are vasoconstrictors
F2-Isoprostanes
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Roberts et al. 5
Intermediates in the IsoP Pathway are
Prostaglandin H2-Like Bicyclic Endoperoxides
•
•
PGH2 is unstable, t½ in aqueous buffer ~5 mins
•
However, we found that the reduction of the
endoperoxides in vivo is not completely efficient
We have demonstrated that GSH is a key effector of
the reduction of IsoP endoperoxides in vivo to F2IsoPs and that other thiols can substitute
– Consequently, the endoperoxides undergo
rearrangement in vivo to form other products
F2-Isoprostanes
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H2-IsoPs Rearrange to Form E2-IsoPs, D2-IsoPs,
and Isothromboxanes In Vivo
R
O
O
R'
H2-IsoP
Reduction
OH
Rearrangement
R
OH
OH
O
R
R
+
+
R'
OH
F2-IsoP
F2-Isoprostanes
R'
R'
OH
E2-IsoP
R
HO
O
R'
O
D2-IsoP
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IsoTxB2
Roberts et al. 7
Are Cyclopentenone PGA2 and PGJ2-Like
IsoPs Formed In Vivo?
Formed by Dehydration of E2- and D2-IsoPs
OH
O
R
R
R'
R'
• Cyclopentenone prostanoids are
reactive compounds
O
OH
E2-IsoP
D2-IsoP
H2 O
H2O
O
R
R
R'
R'
• They undergo Michael addition
reactions with thiols, e.g. GSH, and
covalently bind to proteins
O
A2-IsoP
F2-Isoprostanes
J2-IsoP
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Roberts et al. 8
Cyclopentenone Prostanoids
•
Possess unique biological properties
– Inhibit cellular proliferation due to their ability to
modulate a variety of growth and stress related
genes
>Induce cell cycle arrest and differentiation
>Induce apoptosis
– Activate PPAR-
F2-Isoprostanes
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Cyclopentenone Prostanoids
• Whether cyclooxygenase derived PGA2 or PGJ2 are
actually formed in vivo has been the subject of heated
controversy for over 2 decades
• This may be because their detection in vivo may be
complicated by their sequestration as adducts, e.g. with GSH
• Since IsoPs are initially formed esterified on phospholipids,
A2/J2-IsoPs may be shielded from this process while
esterified in membranes, but would rapidly conjugate with
GSH once released by phospholipase action
F2-Isoprostanes
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Roberts et al. 10
O
O
C
OH
PLase
COOH
O
O
J2-IsoP
OH
GSH
GSH
GSH
GSH
GSH
GSH
NH 2
NHCOCH 2CH 2CHCOOH
CHCONHCH 2COOH
CH 2
S
COOH
O
F2-Isoprostanes
OH
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Roberts et al. 11
Time Course of Conjugation of 15-A2t-IsoP (8-IsoPGA2) with GSH and Adduction to BSA
Conjugation With GSH
Adduction to BSA
60
75
% Adducted
% Conjugated
100
50
25
0
0
4
8
12
Time (min)
F2-Isoprostanes
16
40
20
0
0
25
50
75
100
125
Time (min)
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Roberts et al. 12
Levels of A2/J2- and E2/D2-IsoPs Esterified in Rat Liver
Following Administration of CCl4 to Induce an Oxidant Injury
Liver Esterified
A2/J 2-IsoPs (ng/g) 150
600
100
400
50
0
Liver Esterified
E2/D2-IsoPs (ng/g)
200
-CCl4
+CCl4
A2/J 2-IsoPs
-CCl4
+CCl4
0
E2/D2-IsoPs
Although E2/D2-IsoP levels increase dramatically in plasma after CCl4, A2/J2-IsoPs
could not be detected in plasma even after CCl4, presumably because the free
compounds rapidly and efficiently conjugate with thiols
F2-Isoprostanes
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Roberts et al. 13
Rearrangement of PGH2
O
COOH
PGH2
O
OH
PGE2 & PGD2
O
COOH
LGE2
O
20% of products
• Reactive molecules
OH
• Adduct to proteins
O
COOH
LGD2
O
and form cross-links
OH
Levuglandins
F2-Isoprostanes
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Roberts et al. 14
Are Levuglandin-Like -Ketoaldehydes (Isoketals)
Formed via the IsoP Pathway?
R
O
O
R'
H2-IsoP
Rearrangement
O
O
R
R
+
R'
O
E2-IsoK
F2-Isoprostanes
R'
O
D2-IsoK
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Roberts et al. 15
Detection of the Formation of IsoKs
•
Detected in abundance during oxidation of arachidonic
acid in vitro
•
However, could not be detected during oxidation of
simple biological systems, e.g. LDL or microsomes
– Speculated this may be due to rapid adduction to
proteins
– We therefore compared the rates of adduction of
IsoK and 4-HNE to OVA and their ability to induce
cross-links
F2-Isoprostanes
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Roberts et al. 16
Comparison of the Rate of Adduction to Ovalbumin
and Protein Cross-Linking by IsoKs and 4-HNE
% Free Compounds
Remaining
100
Adduction to OVA
IsoK
HNE
(4 hrs)
80
60
40
20
0
0
10 20 30 40 50 60 70 80
Time (min)
To detect IsoKs in biological systems, we developed a
LC/MS assay for IsoK lysyl lactam adducts after enzymatic
digestion of proteins to individual amino acids
F2-Isoprostanes
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OVA
Roberts et al. 17
Chemistry of IsoK Adduct Formation with Lysine Residues
HOOC
H2O
+Lysine
R
R'
R
R'
O
N
OH
OH
O
NH
Lys
Lys
O
Schiff Base
O
IsoKetal
R
HO
R'
N
OH
Lys
Dihydroxypyrrolidine
2H2O
R'
R
Oxidation
N
Crosslinks
Lys
O2
R
R'
N
F2-Isoprostanes
Pyrrole
2O2
R
O
HO
R'
N
O
Lys
Lys
Lactam
Hydroxylactam
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Roberts et al. 18
IsoKs are Formed In Vivo: Lactam Adduct Levels
in Normal Plasma
Rat
Human
199  78 pg/mL
561  101 pg/mL
n=4
n=6
Normal levels of F2-IsoPs in human plasma: 35  6 pg/mL
F2-Isoprostanes
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Roberts et al. 19
Effect of IsoKs on Proteasome Activity and Viability of
Neuroglial Cells
% Proteasome Activity
100
% Viability
% Control
80
60
40
IC50 = 330 nM
LC50 = 670 nM
20
0
10 -8
10 -7
10 -6
10 -5
E2-IsoK (M)
IsoKs potenly inhibit the proteasome and are highly toxic
F2-Isoprostanes
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Roberts et al. 20
The Entire Isoprostane Pathway
AA
COOH
OOH
OOH
COOH
O
O
O
O
O
COOH
O
COOH
O
O
OOH
OOH
5-Series
COOH
12-Series
8-Series
15-Series
H2-IsoPs
R'
OH
F2-IsoP
R'
O
E2-IsoP
D2-IsoP
O
R
R
R'
R'
A2-IsoP
F2-Isoprostanes
O
O
R'
R'
OH
O
O
J2-IsoP
IsoTxA2
R
R
R
R
R
R
O
OH
O
OH
R'
R'
O
E2-IsoK
O
D2-IsoK
OH
R
HO
O
R'
IsoTxB2
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Roberts et al. 21
Formation of IsoP-Like Compounds
(Neuroprostanes) from Oxidation of DHA
•
Docosahexaenoic acid (DHA) (C22:63) is highly
enriched in brain.
•
Oxidation of DHA forms classes of different compounds
(NeuroPs) analogous to those formed by the IsoP
pathway that may be sensitive biomarkers of oxidant injury
in the brain and participate in mediating oxidant injury
– F4-, E4-, D4-, A4-, J4-NeuroPs and Neuroketals are all
formed and are present at readily detectable levels
in normal human brain
F2-Isoprostanes
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Roberts et al. 22
NeuroP Regioisomers Formed From Abstraction of
Specific bis-Allylic Hydrogen Atoms
C9
C6
COOH
C12
DHA
C15 C18
OH
OH
COOH
C6
OH
OH
OH
OH
COOH
C9
OH
COOH
OH
OH
OH
OH
OH
COOH
C12
COOH
OH
OH
OH
OH
OH
OH
COOH
C15
OH
COOH
OH
OH
OH
COOH
C18
OH
F2-Isoprostanes
OH
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Roberts et al. 23
Levels of F4-NeuroPs and NeuroKs in Brain from
Patients with Alzheimer’s Disease
F4-NeuroPs
*
12
8
4
Controls
AD
*
60
NeuroKs (ng/g)
F4-NeuroPs (ng/g)
16
0
NeuroKs
45
30
15
0
Controls
AD
Both F4-NeuroPs and NeuroKs are significantly increased in AD brain
F2-Isoprostanes
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Roberts et al. 24
Summary
•
•
•
The IsoP and NeuroP pathways form several series of
different compounds
Some compounds such as F-ring and E-ring IsoPs
exert potent receptor dependent biological actions
Other compounds, i.e.IsoKs, NeuroKs, and A2/J2IsoPs, are extremely reactive compounds which are
cytotoxic and exert other biological effects owing to
their ability to adduct to critical biomolecules
F2-Isoprostanes
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Roberts et al. 25