methods to quantify nitric oxide en vivo: concepts and considerations r

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