Methods to Quantify Nitric Oxide en vivo : Concepts and Considerations

1. HOT STUFF! INSTITUTE FOR LOUISIANA STATE BIOLOGY AND MEDICINE FREE RADICAL UNIVERSITY R 12 fl oz (355 mL) Methods to Quantify Nitric Oxide en vivo: Concepts and Considerations

2. Atmospheric Nitrogen Oxides Los Angeles

3. Nitric Oxide • N O Colorless Gas Free Radical Potent Vasodilator (EDRF)

4. N - O H N H N H 3 3 NO Production by Nitric Oxide Synthase + N N H H O H N H N 2 2 2 3 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

5. Nitric Oxide Synthase (NOS) Isoenzymes NOS Constitutive Inducible neuronal NOS (nNOS; NOS 1) inducible NOS (iNOS; NOS 2) endothelial NOS (eNOS; NOS 3)

6. Nitric Oxide is a Pleiotropic Regulator of the Immune, Cardiovascular and Nervous Systems

7. The Chemistry of Nitric Oxide Dictates its Physiological Activity Metal Complexes/Alkyl Radicals Guanylate Cyclase Cytochromes Lipid Radicals IRP-1 Ribonucleotide reductase Direct NO L-Arginine eNOS nNOS iNOS O2 orO2- Indirect RNOS Oxidation Nitration Nitrosation DNA Strand Breaks Lipid Peroxidation Hydroxylation Nitrotyrosine Nitroguanosine Nitrosothiols Nitrosamines

8. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

9. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

10. 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 lightemitted 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?

11. 2NO + O2 2NO2 2NO + 2NO2 2N2O3 2N2O3 + H2O 4NO2- + 2H+ 4NO + O2 + 2H2O 4NO2- + 4H+ Autoxidation of NO

12. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

13. N H N H + 2 H N S O N N 2 3 N-(1-Naphthyl)ethylenediamine Griess Reaction Sulfanilamide +H+ NO2- + NO+ H N S O N H 2 3 2 + H N S O N N N H N H 2 3 2 Azo Dye (max = 540 nm)

14. 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- Although Nitrite is Produced from NO Autoxidation, Nitrate is the Major NO-Derived Metabolite in Plasma and Urine: Role ofHemoglobin

15. 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 268:237-246.

16. 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 +LPS Saline

17. 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).

18. Simultaneous Quantification of Nitrite and Nitrate by HPLC Using the Griess Reaction Rodriguez and Feelisch PNAS 100:336, 2003 Sensitivity: 1 pmol/ml for either anion (injection volume 100µl) No interference by proteins or colored species 100 nM NO2- 100 nM NO3-

19. Sources of NO2-/NO3- in Extracellular Fluids (Blood, Lymph, Urine, Saliva): • NO • Bacteria (Enteric; Oral) • Diet (need for fasting or nitrate/ • nitrite-free chow)

20. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

21. 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

22. 700 4x106 PMNs Grisham et. al, Gastroenterology,1992 600 500 400 Triazole Formation (nM) 2x106 PMNs 300 200 1x106 PMNs 100 0 1 2 3 4 0 Time (hr) N-Nitrosation of DAN By Extravasated PMNs

23. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

24. Bioimaging of Nitric Oxide Using Diaminofluoresceine-2 (DAF-2) DAF-2 T DAF-2 DAF-2 DA N N H N H N H 2 2 N H N H N 2 2 NOx esterases - - C O O O O C O O O - O O O - O O O A c O O O A c 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

25. t = 0 0.5 min 1 min 1.5 min 2 min 5 min Propagation of NO Wave during Stimulation of Endothelial Cells with the Calcium Ionophor, A23187 (1 µM) Feelisch et. al. Unpublished Observations

26. 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

27. RSNOs S-Nitrosoglutathione (GSNO) S-Nitrosoalbumin (AlbSNO) S-Nitrosohemoglobin (HbSNO) Physiological Roles of Nitrosothiols (RSNOs) Potent vasorelaxants. Antiplatelet activity. Antimicrobial activity. Regulation of vasodilation/ oxygenation (hemoglobin). Intermediates in the metabolism of organic nitrites and nitrates. Regulation of cell signaling/ protein functions.

28. NO-Dependent Formation of S-Nitrosothiols (RSNOs): Large Amounts of NO 2NO + O2 2 NO2 2 NO2 + 2 NO 2 N2O3 2 N2O3 + 2 RSH 2 RSNO+ 2 NO2- RSNOs S-Nitrosohemoglobin (SNOHb) S-Nitrosoglutathione (SNOGSH) S-Nitrosoalbumin (SNOAlb)

29. NO-Dependent Formation of S-Nitrosothiols (RSNOs): Physiological Levels of Oxygen, NO and RSH 2NO + O2 2 NO2 2 NO2 + 2 RSH 2 NO2- + 2 RS + 2H+ 2 NO + 2 RS 2 RSNO ● ● RSNOs S-Nitrosohemoglobin (SNOHb) S-Nitrosoglutathione (SNOGSH) S-Nitrosoalbumin (SNOAlb)

30. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

31. RSNO + Hg+2 RS- + Hg+3 + NO+ Saville Reaction

32. Sulfanilamide +NO+ H N S O N H 2 3 2 N H N H + 2 H N S O N N 2 3 N-(1-Naphthyl)ethylenediamine + H N S O N N N H N H 2 3 2 Azo Dye (max = 540 nm) Saville/Griess Reaction + RS- RSNO + Hg+2

33. 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 Assay Fluorescence Assay DAF-2 Bio-imaging ● Spectrophotometric Assays for RSNOs Saville Assay Modified Saville Assay

34. Fluorometric Determination of S-Nitrosothiols (RSNO) H N H 2 N N O NO+ N H 2 N H DAN 2 -H2O RSNO + Hg+2 N N N+ N N H N H 2 NAT

35. SNO-Alb SNO-Hb S-Nitrosothiol Formation in Blood of LPS-Treated Rats * + 1400 Jourd’heuil et al BBRC, 2000 1200 1000 800 S-Nitrosothiol Concentration (nM) * 600 400 200 0 + LPS Control

36. 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

37. 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-

38. Peroxynitrite Nitrates Tyrosine to Yield 3-Nitrotyrosine 3-Nitrotyrosine Tyrosine O H O H O N 2 + ONOO- R R

39. 3-Nitrotyrosine Formation in vivo: Specific Footprint for Peroxynitrite?

40. HOCl + NO2 _ Generation of 3-Nitrotyrosine _ ONOO NO O2 _ NO2 + H+ O H NO2 O N 2 O2 R HNO Hemoproteins (MPO,Mb) + H2O2 + NO2 _

41. 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.)

42. LSU Health Sciences Center Martin Feelisch (NitroMed) Steve Laroux (Harvard) Kamer Kilinc (Ankara Univ) Albany College of Medicine David Jourd’heuil LSU Grambling State University Allen M. Miles National Cancer Institute David A. Wink Mike Espey Katrina Miranda (Univ Arizona)