Skip to main content
SpringerLink
Log in

Determination of In Vivo Nitric Oxide Levels in Animal Tissues Using a Novel Spin Trapping Technology

  • Protocol
  • First Online:
Nitric Oxide

Part of the book series: Methods in Molecular Biology ((MIMB,volume 704))

Abstract

It has been established that microdialysis ensured by the passage of aqueous solutions of Fe3+ complexes with N-methyl-d-glucamine dithiocarbamate (MGD ) through fine dialysis fibers permeable for compounds with molecular weights below 5 kDa. These fibers can be implanted into heart, liver, and kidney tissues, enabling effective binding of Fe3+–MGD complexes to nitric oxide generated in interstitial fluids of narcotized rats in vivo. Subsequent treatment of dialyzate samples (60 μL) with sodium dithionite favors conversion of newly formed diamagnetic NO–Fe3+–MGD complexes into electron paramagnetic resonance-detectable NO–Fe2+–MGD complexes. The basal levels of NO determined from the concentrations of the complexes in the respective tissues are similar (1 μМ). The microdialysis data suggest that treatment of rats with a water-soluble analogue of nitroglycerine or a dinitrosyl iron complex with thiosulfate induces a long-lasting (>1 h) increase in the steady-state level of NO in animal tissues. This novel technology can be used for comparative analyses of production rates of NO and reactive oxygen species when using iron–dithiocarbamate complexes and spin traps for reactive oxygen species, respectively.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution

Subscribe and save

Springer+ Basic
$34.99 /Month
  • Get 10 units per month
  • Download Article/Chapter or eBook
  • 1 Unit = 1 Article or 1 Chapter
  • Cancel anytime
Subscribe now

Buy Now

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

Similar content being viewed by others

References

  1. Vanin, A. F., Mordvintcev, P. I., Kleschyov, A. L. (1985) Appearance of nitric oxide in animal tissues in vivo. Studia Biophys 120, 135–143.

    Google Scholar 

  2. Kubrina, L. N., Caldwell, W. S., Mordvintcev, P. I., Malenkova, I. V., Vanin, A. F. (1992) EPR evidence for nitric oxide production from guanidine nitrogen of L-arginine in animal tissue in vivo. Biochim Biophys Acta 1099, 233–237.

    Article  PubMed  CAS  Google Scholar 

  3. Doi, K., Akaike, T., Horie, H., Noguchi, Y., Fujii, S., Beppu, T., et al. (1996) Excessive production of nitric oxide in rat solid tumor and its implication in rapid tumor growth. Cancer Suppl 77, 1598–1604.

    CAS  Google Scholar 

  4. Vanin, A. F., Huisman, A., van Faassen, E. (2002) Iron dithiocarbamates as spin traps for nitric oxide: pitfalls and successes. Methods Enzymol 359, 27–42.

    Article  PubMed  CAS  Google Scholar 

  5. Vanin, A. F., Liu, X., Samouilov, A., Stukan, R. A., Zweier, J. L. (2000) Redox properties of iron-dithiocarbamates and their nitrosyl derivatives: implications for their use as traps of nitric oxide in biological systems. Biochim Biophys Acta 1474, 365–377.

    Article  PubMed  CAS  Google Scholar 

  6. Sato, S., Tominaga, T., Ohnishi, T., Ohnishi, S. T. (1994) Electron paramagnetic studies of nitric oxide production in the rat brain subjected to ischemia-reperfusion. Brain Res 647, 91–96.

    Article  PubMed  CAS  Google Scholar 

  7. Hooper, D. C., Ohnishi, S. T., Kean, R., Numagami, Y., Dietzschold, B., Koprovski, H. (1995) Local nitric production in viral and autoimmune diseases of central nervous system. Proc Natl Acad Sci USA 92, 5312–5316.

    Article  PubMed  CAS  Google Scholar 

  8. Quaresima, V., Takehara, H., Tsushima, K., Ferrari, M., Utsumi, H. (1996) In vivo detection of mouse liver nitric oxide generation by spin trapping electron paramagnetic resonance spectroscopy. Biochim Biophys Res Comm 221, 729–734.

    Article  CAS  Google Scholar 

  9. Kleschyov, A. L., Mollnau, H., Oelze, M., Meinertz, T., Huang, Y., Harrison, D., et al. (2000) Spin trapping of vascular nitric oxide using colloid Fe(II)- diethylthiocarbamate. Biochim Biophys Res Comm 275, 672–677.

    Article  CAS  Google Scholar 

  10. Muscari, C., Grossi, L., Giordano, E., Ferrari, D., Bonafe, F., Guarnieri, C., et al. (2003) Evaluation of nitric oxide release in the coronary effluent by a novel EPR technique: a study on isolated rat hearts subjected to cold cardioplegia and reperfusion. Life Sci 74, 109–123.

    Article  PubMed  CAS  Google Scholar 

  11. Khoo, J. P., Alp, N. J., Bendall, J. K., Kawashima, S., Yokoyama, M., Zhang, Y.-H., et al. (2004) EPR quantification of vascular nitric oxide in genetically modified mouse models. Nitric Oxide Biol Chem 10, 156–161.

    Article  CAS  Google Scholar 

  12. Charlier, N., Preat, V., Gallez, B. (2006) Evaluation of lipid-based carrier systems and inclusion complexes of diethyldithiocarbamate-iron to trap nitric oxide in biological systems. Magn Res Med 55, 215–218.

    Article  CAS  Google Scholar 

  13. Piehl, L., Capani, F., Facorro, G., Lopez, E. M., Rubin de Celis, E., Pustvorth, C., et al. (2007) Nitric oxide increases in the rat retina after continuous illumination. Brain Res 1156, 112–119.

    Article  PubMed  CAS  Google Scholar 

  14. Ren, J., Fung, P. C. W., Chang, C., Shen, G. H., Lu, G., Chan, F. H. Y., et al. (2007) A comparative ESR study on blood and tissue nitric oxide concentration during renal ischemia-reperfusion injury. Appl Magn Res 32, 243–255.

    Article  CAS  Google Scholar 

  15. Oteki, T., Nagase, S., Shimohata, H., Hirayama, A., Ueda, A., Yokoyama, H., et al. (2008) Nitric oxide protection against adriamycin-induced tubulointerstitial injury. Free Radic Res 42, 154–161.

    Article  PubMed  CAS  Google Scholar 

  16. Pavlica, N. A., Sentjurc, M., Crossley, D. A., Jerin, A., Erzen, D., Zdovc, I., et al. (2009) Single gavage with Porphyromans gingivalis reduces acute systemic nitric oxide response in mice. Oral Microbiol Immunol 23, 435–439.

    Google Scholar 

  17. Jackson, S. K., Thomas, M. P., Smith, S., Madhani, M., Rogers, S. C., James, P. E. (2003) In vivo EPR spectroscopy: biomedical and potential diagnostic applications. Faraday Discuss 126, 103–117.

    Article  Google Scholar 

  18. Sugata, H., Ueno, T., Shimosegawa, T., Yoshimura, T. (2003) Direct detection of nitric oxide and its roles in maintaining gastric mucosal integrity following ethanol-induced injury in rats. Free Radic Res 37, 159–169.

    Article  PubMed  CAS  Google Scholar 

  19. Kuppusamy, P., Ohnishi, S. T., Numagami, Y., Ohnishi, T., Zweirer, J. L. (1995) Three-dimensional imaging of nitric oxide production in the rat brain subjected to ischemia-hypoxia. J Cereb Blood Flow Metab 15, 899–903.

    Article  PubMed  CAS  Google Scholar 

  20. Yoshimura, T., Fujii, S., Yokoyama, H., Kamada, H. (1995) In vivo electron paramagnetic resonance imaging of NO-bound iron complex in a rat head. Chem Lett 4, 309–310.

    Article  Google Scholar 

  21. Hirayama, A., Nagase, S., Ueda, A., Yoh, K., Oteki, T., Obara, M., et al. (2003) Electron paramagnetic resonance imaging of nitric oxide organ distribution in lipopolysaccharide treated mice. Mol Cell Biochem 244, 63–67.

    Article  PubMed  CAS  Google Scholar 

  22. Lai, C.-S., Komarov, A. M. (1994) Spin trapping of nitric oxide produced in vivo in septic-shock mice. FEBS Lett 345, 120–124.

    Article  PubMed  CAS  Google Scholar 

  23. Komarov, A. M., Lai, C.-S. (1995) Detection of nitric oxide production in mice by spin-trapping electron paramagnetic resonance spectroscopy. Biochim Biophys Acta 1272, 29–36.

    PubMed  Google Scholar 

  24. Miyajima, T, Kotake, Y. (1995) Spin trapping agent, phenyl N-tert-butyl-nitrone, inhibits induction of nitric oxide synthase on endotoxin-induced shock in mice. Biochim Biophys Res Commun 215, 114–121.

    Article  CAS  Google Scholar 

  25. Reinke, L. A., Moor, D. R., Kotake, Y. (1995) Hepatic nitric oxide formation: spin trapping detection in biliary efflux. Anal Biochem 243, 8–14.

    Article  Google Scholar 

  26. Zweier, J. L., Wang, P., Kuppusami, P. (1995) Direct measurement of nitric oxide generation in the ischemic heart using electron paramagnetic resonance spectroscopy. J Biol Chem 270, 304–307.

    Article  PubMed  CAS  Google Scholar 

  27. Mikoyan, V. D., Kubrina, L. N., Serezhenkov, V. A., Stukan, R. A., Vanin, A. F. (1997) Complexes of Fe2+ with diethyldithiocarbamate as traps of nitric oxide in animal tissues: comparative studies. Biochim Biophys Acta 1336, 225–234.

    Article  PubMed  CAS  Google Scholar 

  28. Fujii, H., Koscielniak, J., Berliner, L. J. (1997) Determination and characterization of nitric oxide generation in mice by in vivo L-band EPR spectroscopy. Magn Res Med 38, 565–568.

    Article  CAS  Google Scholar 

  29. Clermont G., Lecour S., Vergely C., Zeller M., Perrin C., Maupoil V., et al. (2003) Direct demonstration of nitric oxide formation in organs of rabbits treated by transdermal glyceryl trinitrate using an in vivo spin trapping technique. Fundam Clin Pharmacol 17, 709–715.

    Article  PubMed  CAS  Google Scholar 

  30. Nagano, Ta.nd, Yoshimura, T. (2002) Bioimaging of nitric oxide. Chem Rev 102, 1235–1269.

    Google Scholar 

  31. Vanin, A., Svistunenko, D., Mikoyan, V., Serezhenkov, V., Fryer, M., Baker, N., et al. (2004) Endogenous superoxide production and the nitrite/nitrate ration control the concentration of bioavailable free nitric oxide in leaves. J Biol Chem 279, 24100–24107.

    Article  PubMed  CAS  Google Scholar 

  32. Timoshin, A. A., Drobotova, D. Y., Lakomkin, V. L., Ruuge, E. K., Vanin, A. F. (2008) Estimation of nitric oxide level in vivo by microdialysis with water-soluble iron-N-methyl-D-glucamine dithiocarbamate complexes as NO traps: a novel approach to nitric oxide spin trapping in animal tissues. Nitric Oxide Biol Chem 19, 338–344.

    Article  CAS  Google Scholar 

  33. Shinobu, L. A., Jones, S. G., Jones, M. M. (1984) Sodium N-methyl-D-glucamine dithiocarbamate and cadmium intoxication. Acta Pharmacol Toxicol 54, 189–194.

    Article  CAS  Google Scholar 

  34. Fujii, S., Yoshimura, T., Kamada, H. (1996) Nitric oxide trapping of water-soluble iron(III) complexes with dithiocarbamate derivatives. Chem Lett 9, 785–786.

    Article  Google Scholar 

  35. Tsuchiya, K., Takasugi, M., Minakuchi, K., Fukuzawa, K. (1996) Sensitive quantitation of nitric oxide by EPR spectroscopy. Free Radic Biol Med 21, 733–737.

    Article  PubMed  CAS  Google Scholar 

  36. Vanin, A. F., Liu, X., Samouilov, A., Stukan, R. A., Zweier, J. L. (2000) Redox Properties of iron-dithiocarbamate and their nitrosyl derivatives: implications for their use as of nitric oxide trapping in biological systems. Biochim Biophys Acta 1474, 365–377.

    Article  PubMed  CAS  Google Scholar 

  37. Fujii, S., Kobayashi, K., Tagawa, S., Yoshimura, T. (2000) Reaction of nitric oxide with iron(III) complex of N-(dithiocarboxy)sarcosine: a new type of reductive nitrosylation involving iron (IY) as an intermediate. J Chem Soc Dalton Trans, 3310–3315.

    Google Scholar 

  38. Timoshin, A. A., Orlova, T. R., Ruuge, E. K., Vanin, A. F. (2005) Determination of nitric oxide in mammals using water-soluble iron(III) dithiocarbamate complex. Biofizika (Rus) 50, 537–543.

    CAS  Google Scholar 

  39. Vanin, A. F., Poltorakov, A. P., Mikoyan, V. D., Kubrina, L. N., van Faassen, E. (2006) Why iron-dithiocarbamates ensure detection of nitric oxide in cells and tissues. Nitric Oxide Biol Chem 15, 293–311.

    Article  Google Scholar 

  40. Vanin, A. F., Bevers, L. M., Mikoyan, V. D., Poltorakov, A. P., Kubrina, L. N., van Faassen, E. (2007) Reduction enhances yields of nitric oxide trapping by iron-dithiocarbamate complex in biological systems. Nitric Oxide Biol Chem 16, 71–81.

    Article  CAS  Google Scholar 

  41. Vanin, A. F., Muller, B., Alencar, J. L., Lobysheva, I. I., Nepveu, F., Stoclet, J.-C. (2002) Evidence that intrinsic iron but not intrinsic copper determines S-nitrosothiol decomposition in buffer solution. Nitric Oxide Biol Chem 7, 194–209.

    Article  CAS  Google Scholar 

  42. Timoshin, A. A., Tskitishvili, O. V., Serebryakova, L. I., Kuzmin, A. I., Medvedev, O.S., Ruuge, E. K. (1994) Microdialysis study of ischemia-induced hydroxyl radicals in the canine heart. Experientia 50, 677–679.

    Article  PubMed  CAS  Google Scholar 

  43. Shumaev, K. B., Lankin, V. Z., Ruuge, E. K., Vanin, A. F., Belenkov, Y. N. (2001) The mechanism of inhibition of free-radical oxidation of β-carotene by S-nitrosoglutathione and iron dinitrosyl complexes. Proc Russian Acad Sci 379, 273–275.

    CAS  Google Scholar 

  44. Shumaev, K. B., Gubkin, A. A., Serezhenkov, V. A., Lobysheva, I. I., Kosmachevskaya, O. V., Ruuge, E. K., et al. (2007) Interaction of reactive oxygen and nitrogen species with albumin- and methemoglobin-bound dinitrosyl iron complexes. Nitric Oxide Biol Chem 18, 37–46.

    Article  Google Scholar 

  45. Pisarenko, O. I., Shulzhenko, V. S., Studneva, I. M., Vanin, A. F., Chazov, E. I. (2008) Biochemical mechanisms of dinitrosyl iron complex action on ischemic rat heart. Izvestiya Ross Acad Nauk ser Biol No 1, 1–5.

    Google Scholar 

  46. Pisarenko, O. I., Shulzhenko, V. S., Studneva, I. M., Pelogeikina, I. A., Timoshin, A. A., Vanin, A. F. (2009) Effects of dinitrosyl iron complex with glutathione and its components on ischemic rat heart during reperfusion. Biofizika (Rus) 54, 1081–1087.

    CAS  Google Scholar 

  47. Rosen, G., Btitigan, B. E., Halpern, H. J., Pou, S. (1999) Free Radicals: Biology and Detection by Spin Trapping. Oxford University Press, Oxford, pp. 26–27 and 86–87.

    Google Scholar 

Download references

Acknowledgement

This work was financially supported by the Russian Foundation for Basic Research (grants No 08-04-00665a and 09-04-00886a).

Author information

Authors and Affiliations

  1. Semyonov Institute of Chemical Physics, Russian Academy of Sciences, Moscow, Russia

    Anatoly F. Vanin

  2. Institute of Experimental Cardiology, Russian Cardiology Research-and-Production Complex, Rosmedtechnology Corporation, Moscow, Russia

    Alexander A. Timoshin

Authors
  1. Anatoly F. Vanin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Alexander A. Timoshin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Anatoly F. Vanin or Alexander A. Timoshin .

Editor information

Editors and Affiliations

  1. School of Pharmacy, Queen's University, Belfast, Lisburn Road 97, Belfast, BT9 7BL, United Kingdom

    Helen O. McCarthy

  2. School of Pharmacy, Queen's University, Belfast, Lisburn Road 97, Belfast, BT9 7BL, United Kingdom

    Jonathan A. Coulter

Rights and permissions

Reprints and permissions

Copyright information

© 2011 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Vanin, A.F., Timoshin, A.A. (2011). Determination of In Vivo Nitric Oxide Levels in Animal Tissues Using a Novel Spin Trapping Technology. In: McCarthy, H., Coulter, J. (eds) Nitric Oxide. Methods in Molecular Biology, vol 704. Humana Press. https://doi.org/10.1007/978-1-61737-964-2_11

Download citation

  • DOI: https://doi.org/10.1007/978-1-61737-964-2_11

  • Published:

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-61737-963-5

  • Online ISBN: 978-1-61737-964-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation