Skip to main content
Log in

Circulating and excretory nitrite and nitrate as indicators of nitric oxide synthesis in humans: methods of analysis

European Journal of Clinical Pharmacology Aims and scope Submit manuscript

Abstract

Nitric oxide (NO) is produced in various cells from l-arginine by the catalytical action of NO synthases (NOS). The main metabolic fate of NO includes oxidation to nitrate by oxyhemoglobin in red blood cells and autoxidation to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in plasma, serum, and urine can be used to assess NO synthesis in humans. Circulating nitrite reflects constitutive endothelial NOS activity. Excretory nitrate indicates systemic NO production. Nitrite and nitrate can be measured in plasma, serum, and urine of humans by various analytical methods which are based on different analytical principles. These methods include colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis, and mass spectrometry. The present article gives an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human plasma, serum, and urine in the framework of clinical studies and discusses their significance.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

References

  1. Furchgott RF, Zawadzki JV (1980) The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine. Nature 288:373–376

    Article  PubMed  CAS  Google Scholar 

  2. Palmer RMJ, Ferrige AG, Moncada S (1987) Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524–526

    Article  PubMed  CAS  Google Scholar 

  3. Ignarro LJ, Buga GM, Wood KS, Byrns RE, Chaudhuri G (1987) Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc Natl Acad Sci U S A 84:9265–9269

    Article  PubMed  CAS  Google Scholar 

  4. Palmer RMJ, Rees DD, Ashton DS, Moncada S (1988) l-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation. Biochem Biophys Res Commun 153:1251–1256

    Article  PubMed  CAS  Google Scholar 

  5. Schmidt, HHHW, Nau H, Wittfoht W, Gerlach J, Prescher KE, Klein MM, Niroomand F, Böhme E (1988) Arginine is a physiological precursor of endothelium-derived nitric oxide. Eur J Pharmacol 154:213–216

    Article  PubMed  CAS  Google Scholar 

  6. Hibbs JB Jr, Taintor RR, Vavrin Z (1987) Macrophage cytotoxicity: role for l-arginine deiminase and imino nitrogen oxidation to nitrite. Science 235:473–476

    Article  PubMed  CAS  Google Scholar 

  7. Marletta MA, Yoon PS, Iyengar R, Leaf CD, Wishnok JS (1988) Macrophage oxidation of l-arginine to nitrite and nitrate: nitric oxide is an intermediate. Biochemistry 27:8706–8711

    Article  PubMed  CAS  Google Scholar 

  8. Leaf CD, Wishnok JS, Tannenbaum, SR (1989) l-Arginine is a precursor for nitrate biosynthesis in humans. Biochem Biophys Res Commun 163:1032–1037

    Article  PubMed  CAS  Google Scholar 

  9. Leaf CD, Wishnok JS, Hurley JP, Rosenblad WD, Fox JG, Tannenbaum SR (1990) Nitrate biosynthesis in rats, ferrets and humans. Precursor studies with l-arginine. Carcinogenesis 11:855–858

    Article  PubMed  CAS  Google Scholar 

  10. Hibbs JB Jr, Westenfelder C, Taintor R, Vavrin Z, Kablitz C, Baranowski RL, Ward JH, Menlove RL, McMurry MP, Kushner JP, Samlowski WE (1992) Evidence for cytokine-inducible nitric oxide synthesis from l-arginine in patients receiving interleukin-2 therapy. J Clin Invest 89:867–877

    Article  PubMed  Google Scholar 

  11. Forte P, Copland M, Smith LM, Milne E, Sutherland J, Benjamin N (1997) Basal nitric oxide synthesis in essential hypertension. Lancet 349:837–842

    Article  PubMed  CAS  Google Scholar 

  12. Katz SD, Khan T, Zeballos GA, Mathew L, Potharlanka P, Knecht M, Whelan J (1999) Decreased activity of the l-arginine-nitric oxide metabolic pathway in patients with congestive heart failure. Circulation 99:2113–2117

    PubMed  CAS  Google Scholar 

  13. Förstermann U, Closs EI, Pollock JS, Nakane M, Schwarz P, Gath I, Kleinert H (1994) Nitric oxide synthase isozymes: characterization, purification, molecular cloning, and functions. Hypertension 23:1121–1131

    PubMed  Google Scholar 

  14. Marletta MA (1993) Nitric oxide synthase structure and mechanism. J Biol Chem 268:12231–12234

    PubMed  CAS  Google Scholar 

  15. Andrew PJ, Mayer B (1999) Enzymatic function of nitric oxide synthases. Cardiovasc Res 43:521–531

    Article  PubMed  CAS  Google Scholar 

  16. Vallance P, Leone A, Calver A, Collierr J, Moncada S (1992) Accumulation of an endogenous inhibitor of nitric oxide synthesis in chronic renal failure. Lancet 339:572–575

    Article  PubMed  CAS  Google Scholar 

  17. Leiper J, Vallance P (2003) Biological significance of endogenous methylarginines that inhibit nitric oxide synthases. Cardiovasc Res 43:542–548

    Article  Google Scholar 

  18. Ford PC, Wink DA, Standbury DM (1993) Autoxidation kinetics of aqueous nitric oxide. FEBS Lett 326:1–3

    Article  PubMed  CAS  Google Scholar 

  19. Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM (1992) Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem 267:24173–24176

    PubMed  CAS  Google Scholar 

  20. Xia Y, Zweier JL (1997) Superoxide and peroxynitrite generation from inducible nitric oxide synthase in macrophages. Proc Natl Acad Sci U S A 94:6954–6958

    Article  PubMed  CAS  Google Scholar 

  21. Vásquez-Vivar J, Kalyanaraman B, Martásek P, Hogg N, Masters BSS, Karoui H, Tordo H, Pritchard KA (1998) Superoxide generation by endothelial nitric oxide synthase: the influence of cofactors. Proc Natl Acad Sci U S A 95:9220–9225

    Article  PubMed  Google Scholar 

  22. Huie RE, Padmaja S (1993) The reaction of NO with superoxide. Free Radic Res Commun 18:195–199

    Article  PubMed  CAS  Google Scholar 

  23. Halliwell B, Gutteridge JMC (1999) Free radicals in biology and medicine. Oxford University Press, Oxford

    Google Scholar 

  24. Pfeiffer S, Gorren ACF, Schmidt K, Werner ER, Hansert B, Bole DS, Mayer B (1997) Metabolic fate of peroxynitrite in aqueous solution: reaction with nitric oxide and pH-dependent decomposition to nitrite and oxygen in a 2:1 stoichiometry. J Biol Chem 272:3465–3470

    Article  PubMed  CAS  Google Scholar 

  25. Kissner R, Koppenol W (2002) Product distribution of peroxynitrite decay as a function of pH, temperature, and concentration. J Am Chem Soc 124:234–239

    Article  PubMed  CAS  Google Scholar 

  26. Herold S, Exner M, Nauser T (2001) Kinetic and mechanistic studies of the NO-mediated oxidation of oxymyoglobin and oxyhemoglobin. Biochemistry 40:3385–3395

    Article  PubMed  CAS  Google Scholar 

  27. Wennmalm Å, Benthin G, Petersson AS (1992) Dependence of the metabolism of nitric oxide (NO) in healthy human whole blood on the oxygenation of its red cell haemoglobin. Br J Pharmacol 106:507–508

    PubMed  CAS  Google Scholar 

  28. Wennmalm Å, Benthin G, Edlund A, Jungsten L, Kieler-Jensen N, Lundin S, Westfelt UN, Petersson AS, Waagstein F (1993) Metabolism and excretion of nitric oxide in humans: an experimental and clinical study. Circ Res 73:1121–1127

    PubMed  CAS  Google Scholar 

  29. Tsikas D (2005) Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids. Free Radic Res 39:797–815

    Article  PubMed  CAS  Google Scholar 

  30. Rhodes P, Leone AM, Francis PL, Struthers AD, Moncada S (1995) The l-arginine: nitric oxide pathway is the major source of plasma nitrite in fasted humans. Biochem Biophys Res Commun 209:590–596

    Article  PubMed  CAS  Google Scholar 

  31. Tsikas D (2004) Measurement of nitric oxide synthase activity in vivo and in vitro by gas chromatography-mass spectrometry. Methods Mol Biol 279:81–104

    PubMed  CAS  Google Scholar 

  32. Vallance P, Patton S, Bhagat K, MacAllister R, Radomski M, Moncada S, Malinski T (1995) Direct measurement of nitric oxide in human beings. Lancet 345:153–154

    Article  Google Scholar 

  33. Kelm M, Schrader J (1990) Control of coronary vascular tone by nitric oxide. Circ Res 66:1561–1575

    PubMed  CAS  Google Scholar 

  34. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S (1991) Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 181:852–857

    Article  PubMed  CAS  Google Scholar 

  35. Bode-Böger SM, Böger RH, Löffler M, Tsikas D, Brabant G, Frölich JC (1999) l-arginine stimulates NO-dependent vasodilation in healthy humans: effect of somatostatin pretreatment. J Investig Med 47:43–50

    PubMed  Google Scholar 

  36. Lauer T, Preik M, Rassaf T, Strauer BE, Deussen A, Feelisch M, Kelm M (2001) Plasma nitrite rather than nitrate reflects regional endothelial nitric oxide synthase activity but lacks intrinsic vasodilator action. Proc Natl Acad Sci U S A 98:12814–12819

    Article  PubMed  CAS  Google Scholar 

  37. Kleinbongard P, Dejam A, Lauer T, Rassaf T, Schindler A, Picker O, Scheeren T, Gödecke A, Schrader J, Schulz R, Heusch G, Schaub GA, Bryan NS, Feelisch M, Kelm M (2003) Plasma nitrite reflects constitutive nitric oxide synthase activity in mammals. Free Radic Biol Med 35:790–796

    Article  PubMed  CAS  Google Scholar 

  38. Kanno K, Hirata Y, Emori T, Ohta K, Egush S, Imai T, Marumo F (1992) l-Arginine infusion induces hypotension and diuresis/natriuresis with concomitant increased urinary excretion of nitrite/nitrate and cyclic GMP in humans. Clin Exp Pharmacol Physiol 19:619–625

    Article  PubMed  CAS  Google Scholar 

  39. Bode-Böger SM, Böger RH, Creutzig A, Tsikas D, Gutzki FM, Alexander K, Frölich JC (1994) l-Arginine infusion decreases peripheral resistance and inhibits platelet aggregation in healthy subjects. Clin Sci 87:303–310

    PubMed  Google Scholar 

  40. Borgonio A, Witte K, Stahrenberg R, Lemmer B (1999) Influence of circadian time, ageing, and hypertension on the urinary excretion of nitric oxide metabolites in rats. Mech Ageing Dev 111:23–37

    Article  PubMed  CAS  Google Scholar 

  41. Bode-Böger SM, Böger RH, Kielstein JT, Löffler M, Schäffer J, Frölich JC (2000) Role of endogenous nitric oxide in circadian blood pressure regulation in healthy humans and in patients with hypertension or atherosclerosis. J Investig Med 48:125–132

    PubMed  Google Scholar 

  42. Elherik K, Khan F, McLaren M, Kennedy G, Belch JJF (2002) Circadian variation in vascular tone and endothelial cell function in normal males. Clin Sci 102:547–552

    Article  PubMed  CAS  Google Scholar 

  43. Tsikas D, Böger RH, Bode-Böger SM, Gutzki FM, Frölich JC (1994) Quantification of nitrite and nitrate in human urine and plasma as pentafluorobenzyl derivatives by gas chromatography-mass spectrometry using their 15N-labelled analogs. J Chromatogr B 661:185–191

    Article  CAS  Google Scholar 

  44. Keimer R, Stutzer FK, Tsikas D, Troost R, Gutzki FM, Frölich JC (2003) Lack of oxidative stress during sustained therapy with isosorbide dinitrate and pentaerythrityl tetranitrate in healthy humans: a randomized, double-blind crossover study. J Cardiovasc Pharmacol 41:284–292

    Article  PubMed  CAS  Google Scholar 

  45. Ellis G, Adatia I, Yazdanpanah M, Makela SK (1998) Nitrite and nitrate analyses: a clinical biochemistry perspective. Clin Biochem 31:195–220

    Article  PubMed  CAS  Google Scholar 

  46. Tsikas D (2005) Analysis of the l-arginine/nitric oxide pathway: the unique role of mass spectrometry. Curr Pharmaceut Anal 1:15–30

    Article  CAS  Google Scholar 

  47. Leone AM, Francis PL, Rhodes P, Moncada S (1994) A rapid and simple method for the measurement of nitrite and nitrate in plasma by high-performance capillary electrophoresis. Biochem Biophys Res Commun 200:951–957

    Article  PubMed  CAS  Google Scholar 

  48. Ueda T, Maekawa T, Sadamitsu D, Oshita S, Ogino K, Nakamura K (1995) The determination of nitrite and nitrate in human blood plasma by capillary zone electrophoresis. Electrophoresis 16:1002–1004

    Article  PubMed  CAS  Google Scholar 

  49. Farell AJ, Blake DR, Palmer RMJ, Moncada S (1992) Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases. Ann Rheum Dis 51:1219–1222

    Google Scholar 

  50. Yang BK, Vivas EX, Reiter CD, Gladwin MT (2003) Methodologies for the sensitive and specific measurement of S-nitrosothiols, iron-nitrosyls, and nitrite in biological samples. Free Radic Res 37:1–10

    Article  PubMed  CAS  Google Scholar 

  51. Misko TP, Schilling RJ, Salvemini D, Moore WM, Currie MG (1993) A fluorometric assay for the measurement of nitrite in biological samples. Anal Biochem 214:11–16

    Article  PubMed  CAS  Google Scholar 

  52. Marzinzig M, Nussler AK, Stadler J, Marzinzig E, Barthlen W, Nussler NC, Berger HG, Morris SM, Brückner UB (1997) Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric Oxide 1:177–189

    Article  PubMed  CAS  Google Scholar 

  53. Tesch JW, Rehg WR, Sievers RE (1976) Microdetermination of nitrates and nitrites in saliva, blood, water, and suspended particulates in air by gas chromatography. J Chromatogr 126:743–755

    Article  PubMed  CAS  Google Scholar 

  54. Bode-Böger SM, Böger RH, Schröder EP, Frölich JC (1994) Exercise increases systemic nitric oxide production in men. J Cardiovasc Risk 1:173–178

    PubMed  Google Scholar 

  55. Smythe GA, Matanovic G, Yi D, Duncan MW (1999) Trifluoroacetic anhydride-catalyzed nitration of toluene as an approach to the specific analysis of nitrate by gas chromatography-mass spectrometry. Nitric Oxide 3:67–74

    Article  PubMed  CAS  Google Scholar 

  56. Tsikas D, Gutzki FM, Sandmann J, Schwedhelm E, Frölich JC (1999) Quantification of plasma and urinary nitrate after its reduction to nitrite and to the pentafluorobenzyl derivative by gas chromatography-tandem mass spectrometry. J Chromatogr B 731:285–301

    Article  CAS  Google Scholar 

  57. Tsikas D (2000) Simultaneous derivatization and quantification of the nitric oxide metabolites nitrite and nitrate in biological fluids by gas chromatography-mass spectrometry. Anal Chem 72:4064–4072

    Article  PubMed  CAS  Google Scholar 

  58. Green LC, Wagner DA, Glokowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite and [15N]nitrate in biological fluids. Anal Biochem 126:131–138

    Article  PubMed  CAS  Google Scholar 

  59. Radomski JL, Palmiri C, Heran WL (1978) Concentrations of nitrate in normal human urine and the effect of nitrate ingestion. Toxicol Appl Pharmacol 45:63–68

    Article  PubMed  CAS  Google Scholar 

  60. Moshage H, Kok B, Huizenga JR, Jansen PLM (1995) Nitrite and nitrate determinations in plasma: a critical evaluation. Clin Chem 41:892–896

    PubMed  CAS  Google Scholar 

  61. El Menyawi I, Looareesuwan S, Knapp S, Thalhammer F, Stoiser B, Burgmann H (1998) Measurement of serum nitrite/nitrate concentrations using high-performance liquid chromatography. J Chromatogr B 706:347–351

    Article  CAS  Google Scholar 

  62. Tsikas D, Rossa S, Sandmann J, Frölich JC (1999) High-performance liquid chromatographic analysis of nitrite and nitrate in human plasma as S-nitroso-N-acetylcysteine with ultraviolet absorbance detection. J Chromatogr B 724:199–201

    Article  CAS  Google Scholar 

  63. Tsikas D (2004) Mass spectrometry-validated HPLC method for urinary nitrate. Clin Chem 50:1259–1261

    Article  PubMed  CAS  Google Scholar 

  64. Li H, Meininger CJ, Wu G (2000) Rapid determination of nitrite by reversed-phase high-performance liquid chromatography with fluorescence detection. J Chromatogr B 746:199–207

    Article  CAS  Google Scholar 

  65. Preik-Steinhoff H, Kelm M (1996) Determination of nitrite in human blood by combination of a specific sample preparation with high-performance anion-exchange chromatography and electrochemical detection. J Chromatogr B 685:348–352

    Article  CAS  Google Scholar 

  66. Pinto PCAG, Lima JLFC, de Sousa Saraiva MLMF (2003) Sequential injection analysis of nitrites and nitrates in human serum using nitrate reductase. Clin Chim Acta 337:69–76

    Article  PubMed  CAS  Google Scholar 

  67. Tsikas D, Gutzki FM, Rossa S, Bauer H, Neumann C, Dockendorff K, Sandmann J, Frölich JC (1997) Measurement of nitrite and nitrate in biological fluids by gas chromatography-mass spectrometry and by the Griess assay: problems with the Griess assay—solutions by gas chromatography-mass spectrometry. Anal Biochem 244:208–220

    Article  PubMed  CAS  Google Scholar 

  68. Smith CCT, Stanyer L, Betteridge DJ (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 Chromatogr B 779:201–209

    Article  CAS  Google Scholar 

  69. Ricart-Jané D, Llobera M, López-Tejero MD (2002) Anticoagulants and other preanalytical factors interfere in plasma nitrate/nitrite quantification by the Griess method. Nitric Oxide 6:178–185

    Article  PubMed  CAS  Google Scholar 

  70. Verdon CP, Burton BA, Prior RL (1995) Sample pretreatment with nitrate reductase and glucose-6-phosphate dehydrogenase quantitatively reduces nitrate while avoiding interferences by NADP+ when the Griess reaction is used to assay for nitrite. Anal Biochem 224:502–508

    Article  PubMed  CAS  Google Scholar 

  71. Dunphy MJ, Goble DD, Smith DJ (1990) Nitrate analysis by capillary gas chromatography. Anal Biochem 184:381–387

    Article  PubMed  CAS  Google Scholar 

  72. Gutzki FM, Tsikas D, Alheid U, Frölich JC (1992) Determination of endothelium-derived nitrite/nitrate by gas chromatography/tandem mass spectrometry using (15N)NaNO2 as internal standard. Biol Mass Spectrom 21:97–102

    Article  PubMed  CAS  Google Scholar 

  73. Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1:307–310

    PubMed  CAS  Google Scholar 

  74. Porter AMW (1999) Misuse of correlation and regression in three medical journals. J R Soc Med 92:123–128

    PubMed  CAS  Google Scholar 

  75. Shah VP, Midha KK, Dighe S, McGilveray IJ, Skelly JP, Yacobi A, Layloff T, Viswanathan CT, Cook CE, McDowall RD, Pittmann KA, Spector S (1992) Analytical methods validation—bioavailability, bioequivalence, and pharmacokinetic studies. J Pharm Sci 81:309–312

    Article  Google Scholar 

  76. Becker AJ, Ückert S, Tsikas D, Noack H, Stief CG, Frölich JC, Wolf G, Jonas U (2000) Determination of nitric oxide metabolites by means of the Griess assay and gas chromatography-mass spectrometry in the cavernous and systemic blood of healthy males and patients with erectile dysfunction during different functional conditions of the penis. Urol Res 28:364–369

    Article  PubMed  CAS  Google Scholar 

  77. Heckmann M, Kreuder J, Riechers K, Tsikas D, Boedeker RH, Reiss I, Gortner L (2004) Plasma arginine and urinary nitrate and nitrite excretion in bronchopulmonary dysplasia. Biol Neonate 85:173–178

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors thank Prof. J. C. Frölich, the former Head and Director of the Institute of Clinical Pharmacology, for his invaluable support. The laboratory assistance of B. Beckmann, I. Fuchs, A. Mitschke and M.-T. Suchy is gratefully acknowledged.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Dimitrios Tsikas.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Tsikas, D., Gutzki, FM. & Stichtenoth, D.O. Circulating and excretory nitrite and nitrate as indicators of nitric oxide synthesis in humans: methods of analysis. Eur J Clin Pharmacol 62 (Suppl 1), 51–59 (2006). https://doi.org/10.1007/s00228-005-0020-z

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00228-005-0020-z

Keywords

Navigation