Las Posibilidades contra La Realidad

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La Química Fisiológica de NO

Las Posibilidades contra La Realidad
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Atmospheric Nitrogen Oxides
Los Angeles
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Nitric Oxide
N
O

Colorless Gas Free Radical Potent Vasodilator
(EDRF)
4
NO Production by Nitric Oxide Synthase

N
H
N
H
O
H
N
H
N
2
2
3
2
N
H
N
H
N
H
O2
O2
NO
NADPH
0.5 NADPH
-OOC
-OOC
-OOC
N
H
3
L-Arginine
NG-Hydroxy-L-Arginine
L-Citrulline
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Nitric Oxide Synthase (NOS) Isoenzymes
NOS
Constitutive
Inducible
inducible NOS (iNOS NOS 2)
neuronal NOS (nNOS NOS 1)
endothelial NOS (eNOS NOS 3)

6
Nitric Oxide is a Pleiotropic Regulator of the
Immune, Cardiovascular and Nervous Systems
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The Chemistry of Nitric Oxide Dictates its
Physiological Activity
Metal Complexes/Alkyl Radicals
Guanylate Cyclase Cytochromes C,O,N
Radicals (Lipid Radicals)
Direct
NO
L-Arginine
eNOS nNOS iNOS
O2 or O2-
Indirect
RNOS
Oxidation
Nitration
Nitrosation
DNA Strand Breaks Lipid Peroxidation Hydroxylation
Nitrotyrosine Nitroguanosine
Nitrosothiols Nitrosamines
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How Does One Quantify NO and/or its Metabolites
in vivo?
? You wish to quantify NO and its
oxidized intermediates in complex physiological
media such as extracellular fluids (serum/plasma
saliva urine tears sweat cerebral-spinal
fluid), cells and tissue. ? You do not have
access to sophisticated instrumentation e.g. ESR
spectrometer, Mass spectrometers etc. ? You do
have access to basic spectro-photometric and
analytical equipment.
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Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO (Nitrate/Nitrite) Griess
Assay Fluorescence Assay DAF-2 Bio-imaging ?
Spectrophotometric Assays for RSNOs Saville
Assay Modified Saville Assay
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NO Detection by Gas Phase Chemiluminescence
Detection Principle NO is purged from an
aqueous solution using an innert gas such as Ar
or He and transferred to a mixing chamber where
it reacts with O3 under reduced pressure. NO
O3 NO2 O2 NO2 NO2 h . ? The light
that is emitted by the fraction of excited NO2 on
returning to the ground state (chemiluminescence
640-3000 nm) is measured by photon counting.
fmol-pmol sensitivity
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The Chemistry of Nitric Oxide Dictates its
Physiological Activity
Metal Complexes/Alkyl Radicals
Guanylate Cyclase Cytochromes C,O,N
Radicals (Lipid Radicals)
Direct
NO
L-Arginine
eNOS nNOS iNOS
O2
Indirect
RNOS
Oxidation
Nitration
Nitrosation
DNA Strand Breaks Lipid Peroxidation Hydroxylation
Nitrotyrosine Nitroguanosine
Nitrosothiols Nitrosamines
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Autoxidation of NO
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Although Nitrite is Produced from NO
Autoxidation, Nitrate is the Major NO-Derived
Metabolite in Plasma and Urine Role of
Hemoglobin
14
Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO (Nitrate/Nitrite) Griess
Assay Fluorescence Assay DAF-2 Bio-imaging ?
Spectrophotometric Assays for RSNOs Saville
Assay Modified Saville Assay
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Quantification of NO-Derived NO3- and NO2- in
Extracellular Fluids
NR
NO3- FAD NADPH NO2- NADP

• Nitrate Reductase (NR) Converts all
• NO3- to NO2-
• All unreacted NADPH must be oxidized
• NO2- quantified by Griess Reaction

Grisham, M.B. et.al Quantitation of nitrate
and nitrite in extracellular fluids. Methods
Enzymol 1996 268237-246.
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Griess Reaction
Sulfanilamide
H
NO
NO2-
H
N
S
O
N
H
2
2
3

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LPS Induces Nitric Oxide Production In Vivo

350
Grisham et. al. Meth Enz, 1996
300
250
200
Serum Nitrate and Nitrite (µM)
150
100
50
0
Saline
LPS
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Measurement of NO2-/NO3- in Plasma Using the
Griess Reagent Problems and Considerations
Heparin may produce precipitant upon addition of
Griess Reagent. Addition of protamine sulfate
will remove heparin (Suggestion Use serum or
calcium chelators such as EDTA or
citrate). NO2-/NO3- may be underestimated in
hemolyzed plasma or Serum due to Hb-catalyzed
oxidation of NO2- (Suggestion Ultrafilterplasma
or serum to obtain a low molecular weight
fraction lt 15,000). The presence of some
plasma or serum-associated proteins may inhibit
nitrate reductase (Suggestion Ultrafilter plasma
or serum to obtain a low molecular weight
fraction).
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Simultaneous Quantification of Nitrite and
Nitrate by HPLC Using the Griess Reaction
Rodriguez and Feelisch PNAS 100336, 2003
Sensitivity 1 nM for either anion (injection
volume 100µl) No interference by proteins or
colored species
100 nM NO2- 100 nM NO3-
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Sources of NO2-/NO3- in Extracellular Fluids
(Blood, Lymph, Urine, Saliva)

• NO
• Enteric Bacteria
• Diet (need for fasting or nitrate/
• nitrite-free chow)

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Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO (Nitrate/Nitrite) Griess
Assay Fluorescence Assay DAF-2 Bio-imaging ?
Spectrophotometric Assays for RSNOs Saville
Assay Modified Saville Assay
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N-nitrosation of 2,3 diaminonaphthalene (DAN) to
yield 2,3-naphthotriazole (NAT)
NO2-
H
DAN
H
N
H
2
N
N
O
NO
N
H
2
N
H
2
-H2O
NAT
N
N
N

N
N
H
N
H
lex 375nm lem 415nm
2
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N-Nitrosation of DAN By Extravasated PMNs
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NO2- Detection by Gas Phase Chemiluminescence
Detection Principle NO2- is reduced using
iodide/I2 in glacial acetic acid to yield
NO NO O3 NO2 O2 NO2 NO2 h .
? The light that is emitted by the fraction of
excited NO2 on returning to the ground state
(chemiluminescence 640-3000 nm) is measured by
photon counting. fmol to pmol sensitivity.
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Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO Griess Assay Fluorescence
Assay DAF-2 Bio-imaging ? Spectrophotometric
Assays for RSNOs Saville Assay Modified Saville
Assay
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Bioimaging of Nitric Oxide Using
Diaminofluoresceine-2 (DAF-2)
DAF-2 T
DAF-2
DAF-2 DA
NOx
esterases
fluorescent, Ex 495 nm, Em 515 nm
non-fluorescent
non-fluorescent, cell-permeable
Advantages Sensitivity for NO (5 nM in vitro)
with high temporal and spatial
resolution. No cross-reactivity to
NO2-/NO3- and ONOO-
Kojima et al., Biol.Pharm. Bull. (1997)
Assay limitations Possible interference by
reducing agents and divalent cations,
requires standardized illumination conditions
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Propagation of NO Wave during Stimulation of
Endothelial Cells with the Calcium Ionophor,
A23187 (1 µM)
Feelisch et. al. Unpublished Observations
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NO-Dependent Formation of S-Nitrosothiols (RSNOs)
2 NO O2 2 NO2 2 NO2 2 NO
2 N2O3 2 N2O3 2 RSH 2 RSNO
2 NO2-
RSNOs S-Nitrosohemoglobin (SNOHb) S-Nitrosoglutath
ione (SNOGSH) S-Nitrosoalbumin (SNOAlb)
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NO-Dependent Formation of S-Nitrosothiols
(RSNOs) Physiological Levels of Oxygen, NO and
RSH
2 NO O2 2 NO2 2 NO2 2 RSH
2 NO2- 2 RS 2H 2 NO 2 RS
2 RSNO
?
?
RSNOs S-Nitrosohemoglobin (SNOHb) S-Nitrosoglutath
ione (SNOGSH) S-Nitrosoalbumin (SNOAlb)
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Physiological Roles of Nitrosothiols (RSNOs)
Potent vasorelaxants.
Antiplatelet activity.
Antimicrobial activity.
Regulation of vasodilation/ oxygenation (hemoglobi
n).
Intermediates in the metabolism of organic
nitrites and nitrates.
Regulation of cell signaling/ protein functions.
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Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO (Nitrate/Nitrite) Griess
Assay Fluorescence Assay DAF-2 Bio-imaging ?
Spectrophotometric Assays for RSNOs Saville
Assay Modified Saville Assay
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Saville Reaction
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Saville/Griess Reaction
RSNO Hg2
RS-
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Methods to Detect NO and its Metabolites
? Gas Phase Chemiluminescence for NO ?
Spectrophotometric Assays for Oxidized
Metabolites of NO (Nitrate/Nitrite) Griess
Assay Fluorescence Assay DAF-2 Bio-imaging ?
Spectrophotometric Assays for RSNOs Saville
Assay Modified Saville Assay
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Fluorometric Determination of S-Nitrosothiols
(RSNO)
H
N
H
2
N
N
O
NO
N
H
2
N
H
DAN
2
-H2O
RSNO Hg2
N
N
N
N
N
H
N
H
2
NAT
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S-Nitrosothiol Formation in Blood of LPS-Treated
Rats


1400
Jourdheuil et al BBRC, 2000
1200
1000
800
S-Nitrosothiol Concentration (nM)

600
400
200
0
Control
LPS
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The Chemistry of Nitric Oxide Dictates its
Physiological Activity
Metal Complexes/Alkyl Radicals
Guanylate Cyclase Cytochromes C,O,N
Radicals (Lipid Radicals)
Direct
NO
L-Arginine
eNOS nNOS iNOS
O2-
Indirect
RNOS
Oxidation
Nitration
Nitrosation
DNA Strand Breaks Lipid Peroxidation Hydroxylation
Nitrotyrosine Nitroguanosine
Nitrosothiols Nitrosamines
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Interaction Between Superoxide and Nitric
Oxide Formation of Peroxynitrite/Peroxynitrous
Acid
H
O2- NO ONOO- ONOO- H
ONOOH ONOOH
ONOOH (OH. NO2.) ONOOH
NO3- H Sum O2- NO
NO3-
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Peroxynitrite Nitrates Tyrosine to
Yield 3-Nitrotyrosine
3-Nitrotyrosine
Tyrosine
O
H
O
H
O
N
2
ONOO-
R
R
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3-Nitrotyrosine Formation in vivo Specific
Footprint for Peroxynitrite?
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Generation of 3-Nitrotyrosine
_
ONOO

NO
O2
_
NO2 H
O
H
NO2
O
N
2
O2
R
HNO
Heme, Hemoproteins H2O2 NO2
_
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Myoglobin-Catalyzed Formation of 3-Nitrotyrosine
(3-NT)
250
200
Kilinc and Grisham BBRC, 2001
150
3-Nitrotyrosine Formation (uM)
Mb
100
H2O2 NO2- Tyr 3NT
50
0
0
10
20
30
Time (min.)
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NO-Derived Nitrosamines (RNNOO) Produced in vivo?
50

Rassef et. al. FRBM, 2002
40
30
RSNO, RNNO (nM)
20
10
0
RNNO
RSNO
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Nitrosamine Formation in vivo
Dietary Nitrite/Gastric Acid
Endogenous NO (NOSEnterics)
NO2-
NO
H
O2
RRN-NO
RRNH2 N2O3
Note Primary or Secondary Amines May be
N-Nitrosated
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Nitrosamine Formation May Mediate DNA Damage and
Promote Cellular Mutations by Two Mechanisms
? Alkylation of DNA bases ? Nitrosative
Deamination of DNA Bases
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Nitrosamine-Mediated Alkylation of DNA Bases
Hydroxylation
N-Nitrosodimethylamine
CH2O H2O
CH3N2

C
H
O
3
N
H
N
N
N
H
N
2
O6Methylguanine
Guanine
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Nitrosative Deamination of DNA Bases by
NO-Derived N2O3
ArNHNO
ArNH2 N2O3
ArNHNO H
ArN2 H2O
ArOH N2 H
ArN2 H2O
ArNH2 represents DNA bases containing an
exocyclic amino group Cytosine, methylCytosine,
Guanine or Adenine
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Types of Mutations that Arise From the
Nitrosative Deamination of DNA Bases
Conversion
Type of Mutation
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References for Methods
Grisham, M.B. et al. Quantitation of nitrate
and nitrite in extracellular fluids. Methods
Enzymol 1996 268237-246. Nims, R.W. et al.
Colormetric assays for nitric oxide and nitrogen
oxide species formed from nitric oxide stock
solutions and donor compounds. Methods Enzymol
1996 26893-105. Miles, A.M. et al.
Determination of nitric oxide using fluorescence
spectroscopy. Methods Enzymol 1996
268105-120. Cook, J.A. Convenient colorimetric
and fluorometric assays for S-nitrosothiols. Anal
Biochem. 1996 Jul 1238(2)150-8. Jourdheuil,
D. et al. S-nitrosothiol formation in blood of
lipopolysaccharide-treated rats. Biochem Biophys
Res Commun 2000 27322-26. Jourdheuil, D. et
al. Dynamic state of S-nitrosothiols in human
plasma and whole blood. Free Radic Biol Med
2000 28409-417.
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Geaux Tigers
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LSU Medical Center
Martin Feelisch Steve Laroux Kamer Kilinc
Albany College of Medicine David Jourdheuil
LSU
Grambling State University
Allen M. Miles
National Cancer Institute
David A. Wink Mike Espey Katrina Miranda