Transcript Virtual Free Radical School Measurement of F2-Isoprostanes and Isofurans as Indices of Oxidative Stress Status Jack Roberts , Josh Fessel, and Jason Morrow Vanderbilt.

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-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
F2-Isoprostanes
Josh Fessel
Society For Free Radical Biology and Medicine
Jason Morrow
Roberts et al. 2
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-Isoprostanes
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Roberts et al. 3
“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-Isoprostanes
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Roberts et al. 4
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-Isoprostanes
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Roberts et al. 5
AA
COOH
-H
.
.
COOH
O2
O2
.
OO
OO
COOH
.
COOH
O2
O2
.
COOH
COOH
COOH
COOH
.
O O
.
OO
.
O
COOH
O
.
O
O
COOH
.
O
COOH
O2
O2
O2
OOH
OOH
O
O
COOH
O
O
COOH
O
COOH
.
O
O
O2
COOH
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. 6
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-Isoprostanes
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Roberts et al. 7
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-Isoprostanes
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Roberts et al. 8
An Important Concept Regarding the Formation
and Measurement of F2-IsoPs
•
Initially formed in situ on phospholipids (PL) and then
released by phospholipase action
Oxidation
PL-AA
Lipase
PL-IsoP
Free IsoP
•
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-Isoprostanes
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Roberts et al. 9
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-Isoprostanes
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Roberts et al. 10
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:
CCL4
P-450
CCL3
F2-Isoprostanes
.
LH
L.
O2
LOO .
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Roberts et al. 11
Comparison of Measurements of F2-IsoPs and MDA
In Vitro
MDA
In Vivo
F2-IsoPs
60
3
40
2
20
1
0
0
10
20
30
40
F2-IsoPs (pmol/mg protein)
4
80
0
50
Fold-Increase Above Baseline
5
100
MDA (nmol/mg protein)
100
90
80
70
60
50
4
2
0
F2-IsoPs
SGPT
MDA
Time (min)
The formation of both in vitro correlate well - note
there is ~25,000 times more MDA than F2-IsoPs
F2-Isoprostanes
However, in vivo levels of F2-IsoPs
detected are ~28-fold higher than MDA
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Roberts et al. 12
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-Isoprostanes
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Roberts et al. 13
Plasma ConcentrationsFree
of FF2-IsoPs
in
Young
and
Aged
Rats
-IsoPs
2
Free F2-IsoPs (pg/ml)
1800
42.9-fold
1500
1200
20.3-fold
900
Measurement of F2-IsoPs
unveils profound oxidant
stress in aged rats
600
300
4.3-fold
0
Young
Aged
(3-4 mos)
(22-24 mos)
Roberts LJ, II & Reckelhoff JA BBRC 287:254, 2001
F2-Isoprostanes
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Roberts et al. 14
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-Isoprostanes
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Roberts et al. 15
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-Isoprostanes
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Roberts et al. 16
Metabolic Fate of 15-F2t-IsoP (8-Iso-PGF2) in Humans
OH
COOH
OH
OH
15-F2t-IsoP

-Oxidation
-oxidation
5-Reduction
5
 -reduction
OH
COOH
Major urinary metabolite
OH
A GC/MS assay for F2-IsoP-M has
been developed
OH
2,3-Dinor-5,6-Dihydro-15-F2t-IsoP
(F2-IsoP-M)
(Roberts LJ, II, et. al. J. Biol. Chem :271:20617, 1996)
F2-Isoprostanes
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Roberts et al. 17
Assessments of F2-IsoP Production in Patients With
Polygenic Hypercholesterolemia
Plasma Esterified
165
110
55
90
60
30
0
0
Normals Patients
*
p<0.001
*
1.0
F2-IsoP-M (ng/mg Cr)
Free F2-IsoPs (pg/ml)
220
Urinary F2-IsoP-M
*
120
*
275
Esterified F2-IsoPs (pg/ml)
Plasma Free
0.8
0.6
0.4
0.2
0.0
Normals Patients
* p<0.0001
Normals Hi-Chol
*p<0.0008
The relative increases in F2-IsoPs assessed by all 3 approaches are in close agreement
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Roberts et al. 18
Partial List of Disorders in Which Measurements Of F2-IsoPs
Has Implicated a Pathogenic Role for Oxidative Stress
•
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•
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•
•
•
•
•
•
•
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
F2-Isoprostanes
•
•
•
•
•
•
•
•
•
•
•
•
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
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Roberts et al. 19
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
•
Competing with this is attack of the [C] by molecular O2
[C + O2
COO] to form other products
•
Accordingly, measurement of F2-IsoPs is an insensitive
marker of lipid peroxidation in setting of high O2 tension,
e.g. hyperoxic lung injury
F2-Isoprostanes
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Roberts et al. 20
Attack of the C• in the IsoP Pathway by O2 Leads to the
Formation of “Other Products”
AA
COOH
RH
R
COOH
•
•
O2
COOH
O
O
O
•
COOH
O
•
COOH
O
O
O2
OO
O
•
COOH
O
Other Products
F2-Isoprostanes
O
O
COOH
•
Isoprostanes
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Roberts et al. 21
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-Isoprostanes
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Roberts et al. 22
Formation of Isofuran Regioisomers
AA
COOH
RH
R
COOH
•
O2
•
COOH
O
O
COOH
O
O
O2
OH
HO
OH
OH
COOH
O
OH
HO
O
HO
HO
OH
COOH
O
HO
OH
OH
HO
HO
COOH
OH
COOH
O
O
OH
OH
F2-Isoprostanes
COOH
O
OH
HO
COOH
O
OH
COOH
OH
COOH
O
OH
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OH
Roberts et al. 23
Effect of O2 Tension on the Formation of F2-IsoPs and IsoFs
During Oxidation of Arachidonic Acid In Vitro
g IsoPs & IsoFs/mg AA
1.5
IsoPs
IsoFs
1.2
0.9
0.6
0.3
0.0
1%
21% 100%
1%
21% 100%
Oxygen Tension
The formation of IsoFs becomes highly favored at high concentrations of O2
F2-Isoprostanes
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Roberts et al. 24
The Ratio of IsoFs to F2-IsoPs Correlates with Tissue Oxygenation In Vivo
Ratio IsoFs/IsoPs
3.2
2.4
1.6
1.0
0.8
0.0
Brain
Kidney
Liv er
Levels of IsoFs are higher than IsoPs in highly oxygenated organs
(brain and kidney) but lower in poorly oxygenated organs (liver)
F2-Isoprostanes
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Roberts et al. 25
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-Isoprostanes
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Roberts et al. 26