methods to quantify nitric oxide en vivo: concepts and considerations r
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Methods to Quantify Nitric Oxideen vivo:
Concepts and Considerations
R
Atmospheric Nitrogen Oxides
Los Angeles
Nitric OxideN O•
Colorless Gas
Free Radical
Potent Vasodilator (EDRF)
NH
NH3H2N+
NH3
NH
N-OHH2 N
NH3
NH
OH2N
NH3-OOC -OOC -OOC
+ NO
NO Production by Nitric Oxide Synthase
O2
NADPH
O2
0.5 NADPH
L-Arginine NG-Hydroxy-L-Arginine L-Citrulline
Nitric Oxide Synthase (NOS) Isoenzymes
NOS
Constitutive Inducible
inducible NOS(iNOS; NOS 2)
neuronal NOS(nNOS; NOS 1)
endothelial NOS(eNOS; NOS 3)
Nitric Oxide is a Pleiotropic Regulator of the Immune, Cardiovascular and Nervous Systems
The Chemistry of Nitric OxideDictates its Physiological Activity
Oxidation
RNOS
Guanylate CyclaseCytochromesLipid Radicals
IRP-1Ribonucleotide reductase
O2 or O2-
Direct
Indirect
L-ArginineeNOSnNOSiNOS
NO
Nitrosation DNA Strand BreaksLipid Peroxidation
HydroxylationNitrosothiolsNitrosamines
NitrotyrosineNitroguanosine
Nitration
Metal Complexes/Alkyl Radicals
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
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 emitted by excited NO2 upon returning to the ground state is measured by photon counting (fmol-pmol).Not very useful when attempting to quantify NO in physiological fluids such as serum, plasma or urine. Why?
Autoxidation of NO
2NO + O2 2NO2
2NO + 2NO2 2N2O3
2N2O3 + H2O 4NO2- + 2H+
4NO + O2 + 2H2O 4NO2- + 4H+
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
H2NSO3 N N+
+
2 + NO+H2NSO 3 NH
Sulfanilamide
NH NH2
N-(1-Naphthyl)ethylenediamine
Griess ReactionGriess Reaction
NH NH2NNH2N S O3
Azo Dye (max = 540 nm)
NO2-
+H+
Although Nitrite is Produced from NO Autoxidation, Nitrate is the Major NO-Derived Metabolite in Plasma
and Urine:
Role of Hemoglobin
2Hb-Fe+2O2 + 3NO2- + 2H+ 2Hb-Fe+3 + 3NO3
- + H2O
4Hb-Fe+2O2 + 4NO2- + 4H+ 4Hb-Fe+3 + 4NO3
- + O2 + 2H2O
Hb-Fe+2O2 + NO Hb-Fe+3 + NO3-
Quantification of NO-Derived NO3- and NO2
-
in Extracellular Fluids
NO3- + FAD + NADPH NO2
- + NADP+NR
• 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 268:237-246.
LPS Induces Nitric Oxide ProductionIn Vivo
Ser
um
Nit
rate
an
d N
itri
te (
µM
)
0
50
100
150
200
250
300
350 *
Saline +LPS
Grisham et. al.Meth Enz, 1996
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; < 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).
Simultaneous Quantification of Nitrite and Nitrate by HPLC Using the Griess Reaction
Sensitivity:
1 pmol/ml for either anion (injection volume 100µl)
No interference by proteins or colored species
100 nM NO2-
100 nM NO3-
Rodriguez and FeelischPNAS 100:336, 2003
Sources of NO2-/NO3
- in Extracellular Fluids (Blood, Lymph, Urine, Saliva):
• NO
• Bacteria (Enteric; Oral)
• Diet (need for fasting or nitrate/
nitrite-free chow)
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
N-nitrosation of 2,3 diaminonaphthalene (DAN) to yield 2,3-naphthotriazole (NAT)
NH2
NH2
NH
N
N
H
N
NH2
NO
N
NH2
N+
-H2O
DAN
NAT
NO+
ex= 375nmem= 415nm
NO2-
+H+
N-Nitrosation of DAN By Extravasated PMNs
Time (hr)0 1 2 3 4
Tri
azo
le F
orm
atio
n (
nM
)
0
100
200
300
400
500
600
700
4x106 PMNs
2x106 PMNs
1x106 PMNs
Grisham et. al, Gastroenterology, 1992
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
Bioimaging of Nitric Oxide Using Diaminofluoresceine-2 (DAF-2)
Assay limitations: Possible interference by reducing agents and divalent cations, requires standardized illumination conditions
Advantages: Sensitivity for NO (5 nM in vitro) with high temporal and spatial resolution. No cross-reactivity to NO2
-/NO3- and ONOO-
DAF-2 DA
non-fluorescent,cell-permeable
esterases NOx
DAF-2
non-fluorescent
DAF-2 T
fluorescent,Ex 495 nm, Em 515 nm
Kojima et al., Biol.Pharm. Bull. (1997)
O
NH2
AcO OAc
O
O
H2N
O
O
NH 2
COO-
H2N
-O O
H
O-O
N
N N
O
COO-
Propagation of NO Wave during Stimulation of Endothelial Cells with the Calcium Ionophor, A23187 (1 µM)
Feelisch et. al. Unpublished Observations
t = 0 0.5 min 1 min
1.5 min 2 min 5 min
The Chemistry of Nitric OxideDictates its Physiological Activity
Oxidation
RNOS
Guanylate CyclaseCytochromes
C,O,N Radicals(Lipid Radicals)
O2
Direct
Indirect
L-ArginineeNOSnNOSiNOS
NO
Nitrosation DNA Strand BreaksLipid Peroxidation
HydroxylationNitrosothiolsNitrosamines
NitrotyrosineNitroguanosine
Nitration
Metal Complexes/Alkyl Radicals
Physiological Roles of Nitrosothiols (RSNOs)
Potent vasorelaxants.
Regulation of cell signaling/ protein functions.
Antiplatelet activity.
Intermediates in the metabolism of organic nitritesand nitrates.
Antimicrobial activity.
Regulation of vasodilation/ oxygenation(hemoglobin).
RSNOs
S-Nitrosoglutathione (GSNO)S-Nitrosoalbumin (AlbSNO)S-Nitrosohemoglobin (HbSNO)
NO-Dependent Formation of S-Nitrosothiols (RSNOs): Large Amounts
of NO
2 NO + O2 2 NO2
2 NO2 + 2 NO 2 N2O3
2 N2O3 + 2 RSH 2 RSNO + 2 NO2-
RSNOsS-Nitrosohemoglobin (SNOHb)S-Nitrosoglutathione (SNOGSH)S-Nitrosoalbumin (SNOAlb)
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
RSNOsS-Nitrosohemoglobin (SNOHb)S-Nitrosoglutathione (SNOGSH)S-Nitrosoalbumin (SNOAlb)
●
●
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
Saville Reaction
RSNO + Hg+2 RS- + Hg+3 + NO+
Saville/Griess ReactionSaville/Griess Reaction
2 + NO+
H2NSO3 N N+
+
H2NSO 3 NH
Sulfanilamide
NH NH2
N-(1-Naphthyl)ethylenediamine
NH NH2NNH2NSO 3
Azo Dye (max = 540 nm)
RSNO + Hg+2 + RS-
Methods to Quantify NO and its Metabolites in Extracellular Fluids and/or Tissue
● Gas Phase Chemiluminescence (NO)
● Spectrophotometric Assays for Oxidized Metabolites of NO (Nitrite/Nitrate)
Griess AssayFluorescence AssayDAF-2 Bio-imaging
● Spectrophotometric Assays for RSNOsSaville AssayModified Saville Assay
Fluorometric Determination of S-Nitrosothiols (RSNO)
NH
N
N N
NH2
N+
NH2
NH2
H
N
NH2
NO
-H2O
NO+
RSNO + Hg+2
DAN
NAT
S-Nitrosothiol Formation in Blood of LPS-Treated Rats
*+
*
S-N
itro
so
thio
l Co
nce
ntr
atio
n (
nM
)
0
200
400
600
800
1000
Control + LPS
1200
1400Jourd’heuil et alBBRC, 2000
SNO-Alb
SNO-Hb
The Chemistry of Nitric OxideDictates its Physiological Activity
Oxidation
RNOS
Guanylate CyclaseCytochromes
C,O,N Radicals(Lipid Radicals)
O2-
Direct
Indirect
L-ArginineeNOSnNOSiNOS
NO
Nitrosation DNA Strand BreaksLipid Peroxidation
HydroxylationNitrosothiolsNitrosamines
NitrotyrosineNitroguanosine
Nitration
Metal Complexes/Alkyl Radicals
O2- + NO ONOO-
ONOO- + H+ ONOOH
ONOOH ONOOH* (“OH. + NO2.”)
ONOOH* NO3- + H+
Sum: O2- + NO NO3
-
Interaction Between Superoxide and Nitric Oxide:Formation of Peroxynitrite/Peroxynitrous Acid
+ H+
O H
NO2
R
O H
R
+ ONOO-
Tyrosine 3-Nitrotyrosine
Peroxynitrite Nitrates Tyrosine to Yield3-Nitrotyrosine
3-Nitrotyrosine Formation in vivo:
Specific Footprint for Peroxynitrite?
Hemoproteins (MPO,Mb)+ H2O2 + NO2
R
OH
NO2
Generation of 3-Nitrotyrosine
ONOO_
NO2
HNO
O2
_
NO2 + H+NO
HOCl+
NO2
_
_ O2
Myoglobin-Catalyzed Formation of 3-Nitrotyrosine (3-NT)
0
50
100
150
200
250
0 10 20 30Time (min.)
3-N
itro
tyro
sin
e F
orm
ati
on
(u
M)
Kilinc and Grisham BBRC, 2001
H2O2 + NO2- + Tyr 3NT
Mb
Martin Feelisch (NitroMed)Steve Laroux (Harvard)
Kamer Kilinc (Ankara Univ)
LSU Health Sciences Center
Grambling State UniversityAllen M. Miles
National Cancer InstituteDavid A. Wink
Mike EspeyKatrina Miranda (Univ Arizona)
Albany College of Medicine
David Jourd’heuil LSU