Developed Method Application for Nitrite Ion (NO2¯ ) Analysis of Tib -186 Macrophage Like Cell Lines by Rapid Isocratic HPLC System with High Sensitive Glassy Carbon Electrochemical Detector

Document Type: Research Note

Authors

1 Institute of Biochemistry and Biophysics, University of Tehran, P.O.Box 13145-1384, Tehran, Islamic Republic Of Iran, I.R. IRAN

2 Institute of Biochemistry and Biophysics, University of Tehran, P.O.Box 13145-1384, Tehran, Islamic Republic Of Iran, I.R. IRAN

Abstract

A rapid isocratic method of high performance liquid chromatography system (HPLC) with a glassy carbon working electrode of electrochemical detector is set up for quantitative detection of  trace amount of nitrite ion (NO2¯) in aqueous protein containing cell lysate, cell media, plasma, serum, urine and other body fluids. The solid extraction  reversed phase cartridges (Sep-pak) are used for deproteinizing and purification of the samples. Nitrite ion is the only stable end product of autoxidation of nitric oxide (NO) ; which is a highly reactive paramagnetic molecule produced via the enzymatic conversion of L-arginine to L-citroline. The enzyme involved in this process is the inducible nitric oxide synthase (iNOS), the main isoform of the enzymes in macrophage and macrophage like cell lines such as Raw-264, J774, and Ic-21. Nitrite ion (NO2¯) in nanomolar concentration range is measured by the ECD detector with an amperometric cell, applied voltage of + 800 mV and  Ag-AgCl as the reference electrode. Elusion buffer is 8 mM ammonium chloride containing 25% methanol, flow rate of 1 ml/min and column temperature set at 20° C. The reproducibility of sample preparation and analysis had a coefficient of variance (c.v.) less than 10 % in the cell lysates and cell media of the Tib-186 cell lines. Therefore, this will be a reliable analytical method for the nitrite ion analysis under various conditions of cytokines, LPS, irradiation, or other chemical applications for evaluation of the probable over expression of the inducible nitric oxide synthase ( iNOS ) gene in these type of cells.

Keywords

Main Subjects


[1] Moncada, S., Palmer, R.M.J. and Higgs, E.A., Nitric Oxide: Physiology, Pathophysiology and Pharmaco-logy, Pharmacol, Rev., 43, p. 109 (1991).
[2] Hibbs, J.B., Jr. Synthesis of Nitric Oxide from L-Arginine: A Recently Discovered Pathway Induced by Cytokines with Antitumour and a Imicrobial Activity, Res Immunol, 142, p. 565 (1991).
[3] MacMicking, J., Xie, Q.W. and Nathan, C., Nitric Oxide and Macrophage Function, Annu. Res. Immunol, 15, p. 323 (1997).
[4] Lancaster, J.R. Jr., Simulation of the Diffusion and Reaction of Endogenously Produced Nitric Oxide Proc, Natl. Acad. Sci. USA, 91, p. 8137 (1994).
[5] Vanderkooi, J.M., Wright, W.W. and Erecinska, M., Biochem. Biophys. Acta, 1207, p. 249 (1994).
[6] Kim, Y.M., Bergonia, H.A., Muller, C.,  Pitt,  B.R.,  Watkins, W.D. and Lancaster, J.R., J. Biol. Chem. 270, p. 5710 (1995).
[7] Knowles, R.G., Moncada, S., Biochem. J., 298, p. 249 (1994).
[8] Demiryurek, A.T., Cakici, I., Kanzik, I., Pharmacol. Toxicol., 82, p. 113 (1998).
[9] Demiryurek, A.T., Cakici, I., Kanzik, I., Pharmacol. Toxicol., 82, p. 113 (1998).
[10] Joshi, M.S., Lancaster, J.R., Liu, X. and Ferguson, T.B., Nitric oxide, Biology and chemistry, 5 (6), PP. 561-565 (2001).
[11] Michaeil, P. Murphy, Nitric Oxide and Cell Death, Biochim, Biophys. Acta, 1411, p. 401 (1999).
[12] Waters SEP-PAK Cartridge, Care and Use Manual, PN 11188, Rev 4, september, (1990).
[13] Mauel, J., Defendi, V., Infection and Transformation of Mouse Peritoneal Macrophages by Simian Virus 40, J. EXP. Med., 134, p. 335 (1971).
[14] Mauel, J. and Defendi, V., Infection and Trans-formation of Mouse Peritoneal Mmacrophages by Simian Virus 40, J. EXP. Med., 134, p. 335 (1971) PubMed: 71255451.
[15] Marletta, et. al., Macrophage oxidation of L-Arginine to nitrite and nitrate, Biochemistry, 27, p. 8706 (1988).
[16] Sambrook, J.,  Fritsch,  E.F.  and  Mainatis,  T., Molecular cloning, A laboratory manual, 13,  p. 18 (1998).
[17] Miles, AM., Wink, DA., Cook jc. and Gnsham, MB., Determination of nitric oxide using fluorsence spectroscopy, Methods Enzymology, 268, p. 105 (1996).
[18] Brien, J.F., Mc Laughhin B.E., Chemiuluminescence  head  space-gas  analysis  for determination of nitric oxide formation in biological system, Methods Enzymol, 268, p. 83 (1996).
[19] Nazhat, N.B., et. al., Nitric oxide in human plasma and synovial fluid using reaction of    nitric oxide with 3,5-dibromo-4-nitrobenzene sulphonate, BBA-General Subjects,  1427 (2), pp. 276-286  (1999).
[20] Mesaros, S., Determination of nitric oxide saturated solution by amperometry on modified micro-electrode, Methods Enzymol, 301, p. 160 (1999).
[21] Misko, T.P., Schilling, R.J., Salvemini, D.S. and Moore, W.M., A Fluorometric Assay for the Measur-ment of Nitrite in Biological Samples, Anal. Biochem., 214, p. 11 (1993).
[22] Balcioglu, A. and Watkins, CJ., Use of a Hemo-glubin-trapping Approach in the Determination of Nitric Oxide in Vitro and in Vivo Systems, Neurochem Res., 23 (5), p. 815, May. (1998).
[23] Xiaoping Liu., et. al., Diffusion Limited Reaction of Free Nitric Oxide with Erythrocytes , J. Biol. Chem., 273, p. 18709, Jul. (1998).
[24] Awad, H.H. and Stanbury, D.M, Autoxidation of Nitric Oxide in Aqueous Solution, Int. J. Chem. Kinet. 25, p. 375 (1993).
[25] Griess, j.p., Determination of Nitrite and Nitrate by Griess Reaction, Ber. Deutsch  Chem. Ges., 12, p. 426, (1897).
[26] Green,  L.C.,  wagner, D.A.,  Analysis  of  Nitrate, Nitrite and N15 Nitrate in Biological Fluids, Anal. Biochem., 126, p. 131 (1982).
[27] Marzin zig, M. et. al., Improved Methods to Measure and Products of NO in Biological Fluids, Nitric oxide, 1, p. 177 (1997).