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Nitric oxide production: an easily measurable condition index for vertebrates

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Abstract

Nitric oxide (NO) is a multifunctional signalling molecule, acting as a vasodilator, neurotransmitter, and modulator of inflammatory processes. It also participates in killing parasites, virus-infected cells, and tumor cells by formation of peroxynitrite, one of the most important initiators of the free radical damage. Uncontrolled production of NO can lead to nitrosative stress, causing damages to proteins and DNA and cell injury and death. Determination of NO production in animals is potentially informative for description of individual variation in physiological condition, health state, and work load; however, it’s potential in ecophysiological research has remained almost totally unexplored. Here, we describe application of a simple, precise, and inexpensive spectrophotometric assay for determination of NO production from 5 to 10 μL plasma samples of passerine birds. The method is based on estimation of concentrations of nitrate and nitrite—the stable end products of nitric oxide oxidation. The principle of the assay is reduction of nitrate to nitrite by copper-coated cadmium granules, followed by color development with Griess reagent. NO production in captive greenfinches (Carduelis chloris L.) was significantly repeatable over 6-day period (r = 0.35). Injection of an inflammatory agent phytohemagglutinin into wing web resulted in 21% higher levels of NO production at the third day after treatment as compared to saline-injected birds. These findings, consistently with further evidence from veterinary and biomedical literature, suggest that measuring NO production appears an efficient and robust tool for monitoring individual condition and assessment of the magnitude of innate immune response, pathogenicity of infections, and physical effort. We propose that this assay, which is easily applicable in field studies, has an excellent potential in ecophysiological research, particularly in the rapidly developing fields of immunoecology and conservation physiology.

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References

  • Allen PC (1997) Nitric oxide production during Eimeria tenella infections in chickens. Poultry Sci 76:810–813

    CAS  Google Scholar 

  • Allen PC, Fetterer RH (2000) Effect of Eimeria acervulina infections on plasma L-arginine. Poultry Sci 79:1414–1417

    CAS  Google Scholar 

  • Alonso-Alvarez C, Perez-Rodriguez L, Mateo R, Chastel O, Vinuela J (2008) The oxidation handicap hypothesis and the carotenoid allocation trade-off. J Evol Biol 21:1789–1797

    Article  PubMed  CAS  Google Scholar 

  • Banfi G, Malavazos A, Iorio E, Dolci A, Doneda L, Verna R, Corsi MM (2006) Plasma oxidative stress biomarkers, nitric oxide and heat shock protein 70 in trained elite soccer players. Eur J Appl Physiol 96:483–486

    Article  PubMed  CAS  Google Scholar 

  • Bogdan C, Rollinghoff M, Diefenbach A (2000) Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 12:64–76

    Article  PubMed  CAS  Google Scholar 

  • Bourgeon S, Raclot T, Le Maho Y, Ricquier D, Criscuolo F (2007) Innate immunity, assessed by plasma NO measurements, is not suppressed during the incubation fast in eiders. Dev Comp Immunol 31:720–728

    Article  PubMed  CAS  Google Scholar 

  • Chapman ME, Wideman RF Jr (2006) Evaluation of total plasma nitric oxide concentrations in broilers infused intravenously with sodium nitrite, lipopolysaccharide, aminoguanidine, and sodium nitroprusside. Poultry Sci 85:312–320

    CAS  Google Scholar 

  • Chaturvedi CM, Kumar P (2007) Nitric oxide modulates gonadal and adrenal function in Japanese quail Coturnix coturnix japonica. Gen Comp Endocrinol 151:285–299

    Article  PubMed  CAS  Google Scholar 

  • Chown SL, Gaston KJ (2008) Macrophysiology for a changing world. Proc R Soc B Biol Sci 275:1469–1478

    Article  Google Scholar 

  • Cortas NK, Wakid NW (1990) Determination of inorganic nitrate in serum and urine by a kinetic cadmium-reduction method. Clin Chem 36:1440–1443

    PubMed  CAS  Google Scholar 

  • Crippen TL, Sheffield CL, He H, Lowry VK, Kogut MH (2003) Differential nitric oxide production by chicken immune cells. Dev Comp Immunol 27:603–610

    Article  PubMed  CAS  Google Scholar 

  • Feder ME, Bennett AF, Huey RB (2000) Evolutionary physiology. Annu Rev Ecol Syst 31:315–341

    Article  Google Scholar 

  • Hasselquist D (2007) Comparative immunoecology in birds: hypotheses and tests. J Ornithol 148:571–582

    Article  Google Scholar 

  • Hõrak P, Ots I, Tegelmann L, Møller AP (2000) Health impact of phytohaemagglutinin-induced immune challenge on great tit nestlings. Can J Zool 7:905–910

    Article  Google Scholar 

  • Hõrak P, Saks L, Ots I, Kollist H (2002) Repeatability of condition indices in captive greenfinches (Carduelis chloris). Can J Zool 80:636–643

    Article  Google Scholar 

  • Hõrak P, Saks L, Karu U, Ots I, Surai PF, McGraw KJ (2004) How coccidian parasites affect health and appearance of greenfinches. J Anim Ecol 73:935–947

    Article  Google Scholar 

  • Hõrak P, Saks L, Karu T, Ots I (2006) Host resistance and parasite virulence in greenfinch coccidiosis. J Evol Biol 19:277–288

    Article  PubMed  Google Scholar 

  • Hõrak P, Saks L, Zilmer M, Karu U, Zilmer K (2007) Do dietary antioxidants alleviate the cost of immune activation? An experiment with Greenfinches. Am Nat 170:625–635

    Article  PubMed  Google Scholar 

  • Hüsler BR, Blum JW (2001) Blood plasma response and urinary excretion of nitrite and nitrate in milk-fed calves after oral nitrite and nitrate administration. J Nutr Biochem 12:304–309

    Article  PubMed  Google Scholar 

  • Kaya B, Ünal S, Karabulut AB, Türköz Y (2004) Altered diurnal variation of nitric oxide production in patients with panic disorder. Tohoku J Exp Med 204:147–154

    Article  PubMed  CAS  Google Scholar 

  • Lessells CM (2008) Neuroendocrine control of life histories: what do we need to know to understand the evolution of phenotypic plasticity? Philos Trans R Soc B Biol Sci 363:1589–1598

    Article  Google Scholar 

  • Lessells CM, Boag PT (1987) Unrepeatable repeatabilities: a common mistake. Auk 104:116–121

    Google Scholar 

  • Lillehoj HS, Li G (2004) Nitric oxide production by macrophages stimulated with Coccidia sporozoites, lipopolysaccharide, or interferon-gamma, and its dynamic changes in SC and TK strains of chickens infected with Eimeria tenella. Avian Dis 48:244–253

    Article  PubMed  Google Scholar 

  • Mani AR, Nahavandi A, Moosavi M, Safarinejad R, Dehpour AR (2002) Dual nitric oxide mechanisms of cholestasis-induced bradycardia in the rat. Clin Exp Pharmacol Physiol 29:905–908

    Article  PubMed  CAS  Google Scholar 

  • Martin LB II, Scheuerlein A, Wikelski M (2003) Immune activity elevates energy expenditure of house sparrows: a link between direct and indirect costs? Proc R Soc Lond B Biol Sci 270:153–158

    Article  Google Scholar 

  • Millet S, Bennett J, Lee KA, Hau M, Klasing KC (2007) Quantifying and comparing constitutive immunity across avian species. Dev Comp Immunol 31:188–201

    Article  PubMed  CAS  Google Scholar 

  • Romitelli F, Santini SA, Chierici E, Pitocco D, Tavazzi B, Amorini AM, Lazzarino G, Di Stasio E (2007) Comparison of nitrite/nitrate concentration in human plasma and serum samples measured by the enzymatic batch Griess assay, ion-pairing HPLC and ion-trap GC–MS: the importance of a correct removal of proteins in the Griess assay. J Chromatogr B 851:257–267

    Article  CAS  Google Scholar 

  • Schmid-Hempel P (2003) Variation in immune defence as a question of evolutionary ecology. Proc R Soc Lond B Biol Sci 270:357–366

    Article  Google Scholar 

  • Schmid-Hempel P (2005) Evolutionary ecology of insect immune defenses. Annu Rev Entomol 50:529–551

    Article  PubMed  CAS  Google Scholar 

  • Sheldon BC, Verhulst S (1996) Ecological immunology: costly parasite defences and trade-offs in evolutionary ecology. Trends Ecol Evol 11:317–321

    Article  Google Scholar 

  • Sun J, Zhang X, Broderick M, Fein H (2003) Measurement of nitric oxide production in biological systems by using Griess reaction assay. Sensors 3:276–284

    Article  CAS  Google Scholar 

  • Vajdovich P (2008) Free radicals and antioxidants in inflammatory processes and ischemia-reperfusion injury. Vet Clin North Am Small Anim Pract 38:31–123

    Article  PubMed  Google Scholar 

  • Victor VM, Rocha M, De la Fuente M (2004) Immune cells: free radicals and antioxidants in sepsis. Int Immunopharmacol 4:327–347

    Article  PubMed  CAS  Google Scholar 

  • Wikelski M, Cooke SJ (2006) Conservation physiology. Trends Ecol Evol 21:38–46

    Article  PubMed  Google Scholar 

  • Wu KK (2001) Nitric oxide: synthesis and action. In: Encyclopedia of life sciences http://www.els.net. Wiley, New York

  • Xiao DS, Qian ZM (2000) Plasma nitric oxide and iron concentrations in exercised rats are negatively correlated. Mol Cell Biochem 208:163–166

    Article  PubMed  CAS  Google Scholar 

  • Zera AJ, Harshman LG (2001) The physiology of life history trade-offs in animals. Annu Rev Ecol Syst 32:95–126

    Article  Google Scholar 

  • Zera AJ, Harshman LG, Williams TD (2007) Evolutionary endocrinology: the developing synthesis between endocrinology and evolutionary genetics. Annu Rev Ecol Evol Syst 38:793–817

    Article  Google Scholar 

  • Zhu JJ, Lillehoj HS, Allen PC, Yun CH, Pollock D, Sadjadi M, Emara MG (2000) Analysis of disease resistance-associated parameters in broiler chickens challenged with Eimeria maxima. Poultry Sci 79:619–625

    CAS  Google Scholar 

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Acknowledgments

We thank the Sõrve Bird Observatory for providing facilities and Mati Martinson and Lauri Saks for the help in bird trapping. Stefaan Van Dyck (Kemin Agrifoods Europe) kindly donated OroGlo carotenoid supplement. We thank Vallo Tilgar and Priit Kilgas for allowing us to use their unpublished data. Richard Meitern partly analyzed greenfinch samples and reviewed the literature. Lauri Saks, Ulvi Karu, Marju Männiste, and Diana Osuna helped with the experiments, blood sampling, and bird maintenance. Lauri Saks, George Lozano, and anonymous reviewers provided constructive comments on the manuscript. The study was financed by Estonian Science Foundation grant # 6222 to PH and by the European Union through the European Regional Development Fund (Center of Excellence FIBIR) and the Estonian Ministry of Education and Science (target-financing project number 0180004s09). The study was conducted under the license from the Estonian Ministry of the Environment and the experiments comply with the current laws of Estonian Republic.

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  1. Institute of Ecology and Earth Sciences, Tartu University, Vanemuise 46, 51014, Tartu, Estonia

    Elin Sild & Peeter Hõrak

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  1. Elin Sild
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  2. Peeter Hõrak
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Correspondence to Elin Sild.

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Communicated by J. Graves

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Sild, E., Hõrak, P. Nitric oxide production: an easily measurable condition index for vertebrates. Behav Ecol Sociobiol 63, 959–966 (2009). https://doi.org/10.1007/s00265-009-0710-0

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  • DOI: https://doi.org/10.1007/s00265-009-0710-0

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