Detection of Nitros(yl)ated Metabolites of Nitric Oxide (NO) In Vivo by Gas‐Phase Chemiluminescence Assay

Vijay Dhawan1, David Janero1, James Ellis1

1 NitroMed, Inc., Lexington, Massachusetts
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 12.10
DOI:  10.1002/0471141755.ph1210s35
Online Posting Date:  December, 2006
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There are several methods utilized for detection of nitric oxide (NO) and its various metabolites in biological systems. These include spectrophotometric analysis, high performance liquid chromatography, mass spectrophotometry, electro‐paramagnetic resonance (EPR), and gas‐phase chemiluminscence. Of these, the gas‐phase chemiluminescence technique is perhaps the most sensitive, with a limit of detection in the femtomole range. Furthermore, as there are a number of commercially available NO analyzers, it is possible to directly compare results from different laboratories using this technique. Described in this unit is a group‐specific derivatization approach with gas‐phase chemiluminscence detection to profile NO metabolites in vivo.

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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1:

  • NO gas
  • Carrier gas (nitrogen, helium, or argon)
  • KI/I 2 solution (see recipe)
  • Glacial acetic acid
  • 1 N sodium hydroxide
  • Sodium nitrite solutions (see recipe)
  • 0.05 M potassium ferricyanide in PBS (prepare just before use; do not store)
  • Antifoam agent SE‐15 (Sigma)
  • Heparin
  • Rats of desired strain
  • Isoflurane
  • RBC lysis buffer (see recipe)
  • NEM/EDTA‐containing PBS (see recipe)
  • Acidified sulfanilamide (see recipe)
  • 0.2% mercuric chloride (HgCl 2) solution (see recipe)
  • Calibrated commercial NO analyzer (CLD 88 sp from Eco Medics,
  • Glass purge vessel and trap (Part #: CASM 03296‐02 from Ionics,
  • Circulating water baths, 4°C and 60°C
  • 1‐ml syringes with 23‐G needles
  • 10 to 20 ml syringes with 18‐G needles
  • Sterile surgical instruments (dissecting stand, forceps, surgical scissors, microvascular clips)
  • 15‐ml centrifuge tubes
  • Refrigerated centrifuge
  • 20‐G catheter (VWR Scientific)
  • Peristaltic pump system with multi‐channel (4) pump heads (VWR Scientific, model PD5201)
  • Potter‐Elvehjem homogenizer
  • Chromatogram‐processing software (e.g., Chromprocessor; ACD/Labs,
  • Additional reagents and equipment for protein assay ( appendix 3A)
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Literature Cited

Literature Cited
   Cook, J.A., Kim, S.Y., Teague, D., Krishna, M.C., Pacelli, R., Mitchell, J.B., Vodovotz, Y., Nims, R.W., Christodoulou, D., Miles, A.M., Grisham, M.B., and Wink, D.A. 1996. Convenient colorimetric and fluorometric assays for S‐nitrosthiols. Anal. Biochem. 238:150‐158.
   Dhawan, V., Schwalb, D.J., Shumway, M.J., Warren, M.C., Wexler, R.S., Zemtseva, I.S., Zifcak, B.M. and Janero, D.R. 2005. Selective nitros(yl)ation induced in vivo by a nitric oxide‐donating cyclooxygenase‐2 inhibitor: A NObonomic analysis. Free Rad. Bio. Med. 39:1191‐1207.
   Feelisch, M., Rassaf, T., Mnaimneh, S., Singh, N, Bryan, N.S., Jouradheuil, D., and Kelm, M. 2002. Concomitant S‐ and N‐ and heme‐nitros(yl)ation in biological tissues and fluids: implications for the fate of NO in vivo. FASEB J. 16:1775‐1785.
   Janero, D.R., Bryan, N.S., Saijo, F., Dhawan, V., Schwalb, D.J., Warren, M.C., and Feelisch, M. 2004. Differential nitros(yl)ation of blood and tissue constituents during glyceryl trinitrate biotransformation in vivo. Proc. Natl. Acad. Sci. U.S.A. 101:16958‐16963.
   Kelm, M. 1999. Nitric oxide metabolism and breakdown. Biochem. Biophys. Acta. 1411:273‐289.
   McMurtry, M.S., Kim, D.H., Dinh‐Xuan, T., and Archer, S.L. 2000. Measurement of nitric oxide, nitrite and nitrate using chemilumnescence assay: An update for the year 2000. Analysis 28:455‐465.
   Samouilov, A. and Zweier, J.L. 1998. Development of chemiluminscence‐based methods for specific quantitation of nitrosylated thiols. Anal. Biochem. 258:322‐330.
   Saville, B. 1958. A scheme for colorimetric determination of microgram amount of thiols. Analyst 83:670‐672.
   Schmidt, H.H. and Walter, U. 1994. NO at work. Cell 78:919‐25.
   Smith, C.T., Stanyer, L., and Betteridge, D.J. 2002. Evaluation of methods for the extraction of nitrite and nitrate in biological fluids employing high‐performance anion‐exchange liquid chromatography for their determination. J. Chorm. B. 779:201‐209.
   Tsikas, D., Gutzki, F., Rossa, S., Bauer, H., Neumann, C., Dockendorff, K., Sandmann, J., and Frolich, J.C. 1997. Measurement of nitrite and nitrate in biological fluids by gas chromatography‐mass spectrometry and by the Griess assayL: Problems with the Griess assay – solutions by gas chromatography‐mass spectrometry. Anal. Biochem. 244:208‐220.
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