In vivo Detection of LipidDerived Free Radicals

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In vivo Detection of Lipid-Derived Free
Radicals Keizo Sato, M.D., Ph.D. Department of
Respiratory Medicine and Pharmacology/Therapeutics
, Graduate School of Medical and Pharmaceutical
Sciences, Kumamoto University, Honjo 1-1-1,
Kumamoto 860-8556, Japan e-mail
keizokun_at_gpo.kumamoto-u.ac.jp
12th Annual Meeting SFRBM, Austin Texas, Nov.
16-20, 2005
Bacteria
IN VIVO
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POBN-L
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POBN IP
ESR measurement
Keizo Sato
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Kumamoto, Japan
KEIZO SATO MD, PhD Associate Professor Department
of Respiratory Medicine/ Pharmacology
Therapeutics, Graduate School of Medical and
Pharmaceutical Sciences, Kumamoto
University, Kumamoto 860-8556, Japan
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Our problem
Free radical might be produced!!! in various
diseases. No in vivo direct evidence of Free
radical production
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Therefore We focused on the method for
in vivo direct detection of free
radical!!! Using ESR with POBN as a spin trap.
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Todays presentation program

• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

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• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

7
Can we detect the radical?
In vivo direct detection of free radical in
various system. LPS-induced lung injury model
P. aeruginosa-induced pneumonia Diesel
exhaust particle induced lung injury Bleomycin-in
duced lung injury
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LPS P. aeruginosa DEP Bleomycin
Lung injury
Free radical
Measurement By EPR with POBN
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Is it possible to detect directly any free
radicals in LPS -induced ARDS model?
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In vivo ESR detection of lipid-derived radicals
in rat lung treated with intratracheal LPS
instillation
(A) ESR spectrum of POBN radical adducts detected
in lipid extracts of lung 6 h after
intratracheal instillation of LPS and 1 h after
intraperitoneal administration of POBN. (B) Same
as in (A), but rats were not instilled with LPS.
(C) Same as in (A), but rats were not
administrated POBN. (D) Same as in (A), but rats
were not instilled with LPS and were not
administrated POBN.
Sato, K et al. FASEB J 16, 1713-1720, 2002
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Intensity of ESR signals from rat lung treated
with LPS
P 0.023
2.0
Intensity (cm)
1.0
0
LPS-IT Group (n7)
Saline-IT Group (n6)
IT intratracheal instillation
There were markedly increases in the intensities
of the signals after intratracheal LPS
instillation. Intensity mean standard
deviation.
Sato, K et al. FASEB J 16, 1713-1720, 2002
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How about the simulation of this spectrum? What
kind of free radical was detected?
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Computer simulation of the spectrum derived from
rat lung treated with LPS
A. EXPERIMENTAL SPECTRA
B. SIMULATIVE SPECTRA
aHb
aHb
aHb
aN
aN
aN 14.940.07 G, aHb 2.420.06 G
(A) ESR spectra of radical adduct detected in
lipid extract of lung 6 h after intratracheal
instillation of LPS and 1 h after intraperitoneal
administration of 4-POBN. (B) Computer
simulation of the spectrum in (A). The ESR
spectral simulations were performed using an
automatic optimization procedure.
Sato, K et al. FASEB J 16, 1713-1720, 2002
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Comparison with published HFC
C aN 14.940.07 G, aHb 2.420.06 G
Simulation

Lipid-derived free radical (LDFR)
HFC hyperfine coupling constants
There were only minor variations between our
detected radical and published HFC.
Sato, K et al. FASEB J 16, 1713-1720, 2002
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Is this free radical produced in vivo? Is there
no ex vivo production artificially?
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Controls for confirmation of in vivo radical
detection
A. LPS-IT RAT LUNG POBN (EX VIVO)
B. POBN IP LPS (EX VIVO)
C. NON-TREATED LUNG POBN (EX VIVO) LPS (EX
VIVO)
D. NO-LUNG POBN LPS
There were not significant ESR signal observed in
a series of controls for confirmation of in vivo
radical production. (A) ESR spectra of rat lungs
treated with intratracheal LPS instillation and
ex vivo addition of POBN. (B) ESR spectra of
non-treated rat lungs and 1 h after
intraperitoneal administration of POBN and ex
vivo addition of LPS. (C) ESR spectra of
non-treated rat lungs and ex vivo addition of
POBN and LPS. (D) ESR spectra of in vitro
addition of POBN and LPS without rat lung.
Sato, K et al. FASEB J 16, 1713-1720, 2002
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Is it possible to detect directly any free
radicals in P. aeruginosa -induced pneumonia
model?
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In vivo detection of lipid-derived free radicals
in rat lung treated with Pseudomonas aeruginosa
instillation
A. P. aeruginosa -IT RAT LUNG
B. SALINE-IT RAT LUNG
C. INTENSITY OF POBN SPIN ADDUCT
Control Group (n8)
p lt 0.01
P. aeruginosa - IT Group (n7)
Intensity (cm)
Keizo Sato
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Is it possible to detect directly any free
radicals in DEP -induced lung injury model?
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Diesel Exhaust Particles (DEP)
Relative Intensity
LPS priming effect
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Is it possible to detect directly any free
radicals in bleomycin -induced lung injury
model?
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Bleomycin
Vehicle Bleo-IT Bleo-IT Desferal
Relative Intensity
0 5 10 15 20
Vehicle Bleo-IT Bleo-IT Desferal
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About half!!!
EZU Lake in Kumamoto, Japan
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• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

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Chelators DTPA EDTA BC OP NTA Desferal Dipyridil
Organic solvents Phenol Benzene CHCl3 CH3OH CH3OH/
CHCl3
Spin traps DMPO MNP DBNBS PBN POBN PTIO
etc. Radical species O2- OH NO S SOO
SO RO ROO R
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Measurement procedure
Homogenate buffer 5 ml CH3ClCH3OH (21) 1 ml
30 mM 2.2-dipyridyl 4 ml deionized water 4 ml
1.2 mM Phenol ESR condition Bruker EMX Super
high q cavity Freq. 9.79 GHz Field 349540 G
(1024 points) Power 20.2 mW Modulation 100
kHz Mod. Amp. 1.00 G Conversion 655.36 ms
Time constant 1310.72 ms Sweep Time 671.089 s
Homogenize rat lung (35 g) with homogenate
buffer at middle power for 30 sec on ice
bath ? Add 2636 ml CH3ClCH3OH (21)
? Centrifuge at 2,000 rpm for 10 min ? Isolate
the CH3Cl layer (lower) and dry by passing
through sodium sulfate column ? Evaporate the
sample until 1 ml by N2 bubbling ? Record ESR
spectra immediately at room temperature
POBN Alexis
Sato, K et al. FASEB J 16, 1713-1720, 2002
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• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

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Artificial radical production by homogenization
Point Within 30 sec.
Relative intensity (mm)
Homogenizing Time (sec)
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Recrystalization of POBN
A Company native POBN 50 mg/ml in D.W. (other
comp. almost same except for AX company.)
Re-crystalized POBN in 50 mg/ml in D.W.
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Important techniques for handling the sample.
Homogenizing time below 30 sec. Need to use
adequate POBN. Caution about powder from
gloves. Two or more chelators in the sample
can make radical. Anesthetizing agent makes
radical (ketamine etc.)
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Animal experiments POBN ? Organs
extract/homogenize ? Free Radical extraction i.p.
(Lung etc.)
? Stock!!!(CHCl3/CH3OH) -4?
48h -20? 5days -80? 2W
? ESR measurement
Storage!!!
Spin adduct of Lipid-derived free radical is very
stable. So it is possible to measure stocked
sample.
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If you have questions
Welcome to the Workshop Poster Session
305 pm 430 pm
First 10 persons can get Japanese Snack!!! (JOKE)
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Volcano Aso
Dont Sleep!!!
Mt. Aso in Kumamoto, Japan
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• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

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What is the Mechanism of Free Radical
Production?
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?
?
?
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Possible pathway to produce lipid-derived free
radical

• Phagocyte-derived NADPH oxidase dependent pathway
  
• Xanthine oxidase pathway
• 3) NO from iNOS and peroxynitrite pathway

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Hypothetical Pathway
PA
Epithelial Cell
GdCl3
Alveolar Macrophages
Lung
Cytokines
PA Pseudomonas aeruginosa
Neutrophil
NADPH oxidase
NO
NADPH Oxidase KO mice
iNOS KO mice
O2-
Endothelial Cell
Desferal
Metal
Xanthine oxidase
OH
Allopurinol
Lung Injury
L
ONOO-
Keizo Sato
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Mechanism of free radical production in this
model using iNOS KO mice, NADPH oxidase KO mice,
allopurinol (ALP), and desferal (DFO)
plt0.01
plt0.01
plt0.01
P0.06
inhibition of free radical production
PA DFO
PA NADPH Ox KO
PA ALP
PA
PA iNOS KO
Keizo Sato
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Effect of modulating agents/KO mice on P.
aeruginosa (PA) infected lung in histological
findings (x200)
PA-IT
iNOS KO
NADPH OX KO
Allopurinol
Desferal
Keizo Sato
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Summary
Effective
Non-Effective
Cytokines
in PA pneumonia
GdCl3
Epithelial Cell
Alveolar Macrophages
Cytokines
Cytokines
Neutrophil
NADPH oxidase
NO
NADPH Oxidase KO mice
iNOS KO mice
O2-
Endothelial Cell
Desferal
Metal
Xanthine oxidase
OH
Allopurinol
Lung Injury
L
ONOO-
Free radicals were mainly produced by NADPH
oxidase of phagocytes, xanthine oxidase system,
and iron-catalyzed Fenton type reaction.
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Summary2
LPS DEP PA iNOS KO x x
x NADPH ox KO ? ? ?
Allopurinol x ? ? Desferal
? ? ?
We found that mechanism of lipid-derived free
radical production was different depending on
inducing agents.
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• Can we detect the radical?
• Method for the detection of free radical.
• Important techniques for handling the sample.
• Mechanism of lipid-derived free radical
  production.
• Is it possible to use these methods for humans?

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New approach (Preliminary data)
Is it possible to use these methods for humans?
For Humans
Possibility Lipid-derived free radical production
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Lipid-derived free radical production potential
POBN
Free radical production Phagocyte Xanthine
oxidase etc. Antioxidant GSH system Ascorbic acid
system Tocopherol system etc.
Whole blood or BALF
Evaluate the balance of free radical in vivo
Keizo Sato
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Pseudomonas infected mouse
From homogenate
RI 2.0 1.0
PA-IT Cont.
From whole blood
POBN i.p.()
RI 2.0 1.0
PA-IT Cont.
POBN i.p.(-)
Free radical production potential in whole blood
RI 2.0 1.0
Is it possible to detect free radicals in blood
after addition of POBN to the blood?
Possible!!!
PA-IT Cont.
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Lipid-derived free radicals in whole human blood
POBN
LPS
RI 2.0 1.0
Possible!!!
3h incubation at 37?
Whole blood
LPS()
LPS(-)
It is possible to detect from human blood!
Keizo Sato
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Is it possible to detect directly lipid-derived
free radical production potential in human
pneumonia?
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Lipid-derived free radical production potential
in pneumonia patient
82 Male ARDS
mPSL 125mg
POBN
PIPC
Patient blood
Quick response LDFR gt WBCCRP
Keizo Sato
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Conclusion
In various lung injury models, we could detect
free radicals using ESR spin trapping method with
POBN. Using this method we could evaluate the
mechanism of lipid-derived free radicals in
various lung injury models. We have demonstrated
lipid-derived free radical production in human
blood.
Keizo Sato
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Congratulations!!!
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In the Future Study of pathogenesis via in vivo
free radical production. Evaluation of severity
of disease in clinical patients and the
development of new therapeutic agents.
My speciality is lung disease
so I need to continue study of free radical!!!
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Thank you !
Free Radical Metabolite Section, Laboratory of
Pharmacology and Chemistry, National Institute of
Environmental Health Sciences, National
Institutes of Health
Ronald P. Mason Maria B. Kadiiska Toyoko
Arimoto Jean Corbett
Department of Respiratory Medicine, Graduate
School of Medical and Pharmaceutical Sciences,
Kumamoto University, Japan
Satsuki Chibana Junji Nagano Kodai Kawamura
Yasumasa Tashiro Akihisa Yamashita
Hirotsugu Kohrogi Tatsuya Okamoto Hidenori
Ichiyasu Shinichiro Okamoto
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The END
If you have questions
Welcome to the Workshop Poster Session
305 pm 430 pm
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Mechanism?
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Many investigators propose free radicals play
crucial roles in the pathogenesis of many kinds
of diseases. However, it is very difficult to
prove in vivo free radical production. We would
like to present our method and experimental data
of in vivo detection of lipid-derived free
radicals using the electron spin resonance (ESR)
spin-trapping technique. First, we describe
our method. Second, we show in vivo detection of
lipid-derived free radicals in various lung
disease models such as lipopolysaccaride-induced
lung injury, Pseudomonas aeruginosa-induced
pneumonia, bleomycin-induced lung injury, diesel
exhaust-induced lung injury etc. (The FASEB J.
16, 1713-1720, 2002 Am. J. Respir. Crit. Care
Med. 171, 379-87, 2005) Third, we investigate
the mechanism of lipid-derived free radical
production using two types of knockout mice
(NADPH oxidase and iNOS knockout mice) and
modulating agents such as gadolinium chloride,
PMN antibody, cyclophosphamide, desferrioxamine,
uric acid, allopurinol etc. In conclusion, we
discuss the possibilities of this method to
contribute to the development of therapeutic
agents for disease treatment.
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Study the mechanism for lipid-derived free
radical production in P. aeruginosa-induced
pneumonia model.