Analytical and Bioanalytical Chemistry

, Volume 409, Issue 19, pp 4529–4538 | Cite as

Microchip electrophoresis with laser-induced fluorescence detection for the determination of the ratio of nitric oxide to superoxide production in macrophages during inflammation

  • Giuseppe Caruso
  • Claudia G. Fresta
  • Joseph M. Siegel
  • Manjula B. Wijesinghe
  • Susan M. Lunte
Paper in Forefront


It is well known that excessive production of reactive oxygen and nitrogen species is linked to the development of oxidative stress-driven disorders. In particular, nitric oxide (NO) and superoxide (O2 •−) play critical roles in many physiological and pathological processes. This article reports the use of 4-amino-5-methylamino-2′,7′-difluorofluorescein diacetate and MitoSOX Red in conjunction with microchip electrophoresis and laser-induced fluorescence detection for the simultaneous detection of NO and O2 •− in RAW 264.7 macrophage cell lysates following different stimulation procedures. Cell stimulations were performed in the presence and absence of cytosolic (diethyldithiocarbamate) and mitochondrial (2-methoxyestradiol) superoxide dismutase (SOD) inhibitors. The NO/O2 •− ratios in macrophage cell lysates under physiological and proinflammatory conditions were determined. The NO/O2 •− ratios were 0.60 ± 0.07 for unstimulated cells pretreated with SOD inhibitors, 1.08 ± 0.06 for unstimulated cells in the absence of SOD inhibitors, and 3.14 ± 0.13 for stimulated cells. The effect of carnosine (antioxidant) or Ca2+ (intracellular messenger) on the NO/O2 •− ratio was also investigated.

Graphical Abstract

Simultaneous detection of nitric oxide and superoxide in macrophage cell lysates


Bioanalytical methods Inflammation Macrophages Microchip electrophoresis Nitric oxide Superoxide 



This study was funded by the National Science Foundation (CHE-1411993) and the National Institutes of Health (COBRE P20GM103638). G.C. gratefully acknowledges the support of an American Heart Association–Midwest Affiliate Postdoctoral Research Fellowship (NFP0075515). J.M.S. acknowledges the Madison and Lila Self Graduate Fellowship for support. We also thank Richard Piffer and Nancy Harmony for their helpful comments and editorial support.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving human participants and/or animals

This article does not contain any studies with human participants performed by any of the authors.

Supplementary material

216_2017_401_MOESM1_ESM.pdf (211 kb)
ESM 1 (PDF 211 kb)


  1. 1.
    Aaltoma SH, Lipponen PK, Kosma VM. Inducible nitric oxide synthase (iNOS) expression and its prognostic value in prostate cancer. Anticancer Res. 2001;21:3101–6.Google Scholar
  2. 2.
    Patel RP, McAndrew J, Sellak H, White CR, Jo H, Freeman BA, et al. Biological aspects of reactive nitrogen species. Biochim Biophys Acta. 1999;1411:385–400.CrossRefGoogle Scholar
  3. 3.
    Weidinger A, Kozlov AV. Biological activities of reactive oxygen and nitrogen species: oxidative stress versus signal transduction. Biomolecules. 2015;5:472–84.CrossRefGoogle Scholar
  4. 4.
    Li J, Wuliji O, Li W, Jiang ZG, Ghanbari HA. Oxidative stress and neurodegenerative disorders. Int J Mol Sci. 2013;14:24438–75.CrossRefGoogle Scholar
  5. 5.
    Fearon IM, Faux SP. Oxidative stress and cardiovascular disease: novel tools give (free) radical insight. J Mol Cell Cardiol. 2009;47:372–81.CrossRefGoogle Scholar
  6. 6.
    Sosa V, Moline T, Somoza R, Paciucci R, Kondoh H, LLeonart ME. Oxidative stress and cancer: an overview. Ageing Res Rev. 2013;12:376–90.CrossRefGoogle Scholar
  7. 7.
    Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87:315–424.CrossRefGoogle Scholar
  8. 8.
    Stevanin TM, Laver JR, Poole RK, Moir JW, Read RC. Metabolism of nitric oxide by Neisseria meningitidis modifies release of NO-regulated cytokines and chemokines by human macrophages. Microbes Infect. 2007;9:981–7.CrossRefGoogle Scholar
  9. 9.
    Bogdan C. Nitric oxide and the immune response. Nat Immunol. 2001;2:907–16.CrossRefGoogle Scholar
  10. 10.
    Cooke JP. The pivotal role of nitric oxide for vascular health. Can J Cardiol. 2004;20(Suppl B):7B–15.Google Scholar
  11. 11.
    Bredt DS, Hwang PM, Snyder SH. Localization of nitric oxide synthase indicating a neural role for nitric oxide. Nature. 1990;347:768–70.CrossRefGoogle Scholar
  12. 12.
    Ma ZA, Zhao Z, Turk J. Mitochondrial dysfunction and β-cell failure in type 2 diabetes mellitus. Exp Diabetes Res. 2012;2012:703538.CrossRefGoogle Scholar
  13. 13.
    Estevez AG, Jordan J. Nitric oxide and superoxide, a deadly cocktail. Ann N Y Acad Sci. 2002;962:207–11.CrossRefGoogle Scholar
  14. 14.
    Ferrer-Sueta G, Radi R. Chemical biology of peroxynitrite: kinetics, diffusion, and radicals. ACS Chem Biol. 2009;4:161–77.CrossRefGoogle Scholar
  15. 15.
    Szabo C, Ischiropoulos H, Radi R. Peroxynitrite: biochemistry, pathophysiology and development of therapeutics. Nat Rev Drug Discov. 2007;6:662–80.CrossRefGoogle Scholar
  16. 16.
    Espey MG, Miranda KM, Feelisch M, Fukuto J, Grisham MB, Vitek MP, et al. Mechanisms of cell death governed by the balance between nitrosative and oxidative stress. Ann N Y Acad Sci. 2000;899:209–21.CrossRefGoogle Scholar
  17. 17.
    Chi DS, Qui M, Krishnaswamy G, Li C, Stone W. Regulation of nitric oxide production from macrophages by lipopolysaccharide and catecholamines. Nitric Oxide. 2003;8:127–32.CrossRefGoogle Scholar
  18. 18.
    Panaro MA, Brandonisio O, Acquafredda A, Sisto M, Mitolo V. Evidences for iNOS expression and nitric oxide production in the human macrophages. Curr Drug Targets Immune Endocr Metabol Disord. 2003;3:210–21.CrossRefGoogle Scholar
  19. 19.
    Seminara AR, Ruvolo PP, Murad F. LPS/IFNgamma-induced RAW 264.7 apoptosis is regulated by both nitric oxide-dependent and -independent pathways involving JNK and the Bcl-2 family. Cell Cycle. 2007;6:1772–8.CrossRefGoogle Scholar
  20. 20.
    Abbas K, Hardy M, Poulhes F, Karoui H, Tordo P, Ouari O, et al. Detection of superoxide production in stimulated and unstimulated living cells using new cyclic nitrone spin traps. Free Radic Biol Med. 2014;71:281–90.CrossRefGoogle Scholar
  21. 21.
    Li H, Li Q, Wang X, Xu K, Chen Z, Gong X, et al. Simultaneous determination of superoxide and hydrogen peroxide in macrophage RAW 264.7 cell extracts by microchip electrophoresis with laser-induced fluorescence detection. Anal Chem. 2009;81:2193–8.CrossRefGoogle Scholar
  22. 22.
    Mainz ER, Gunasekara DB, Caruso G, Jensen DT, Hulvey MK, da Silva JAF, et al. Monitoring intracellular nitric oxide production using microchip electrophoresis and laser-induced fluorescence detection. Anal Methods. 2012;4:414–20.CrossRefGoogle Scholar
  23. 23.
    de Campos RP, Siegel JM, Fresta CG, Caruso G, da Silva JA, Lunte SM. Indirect detection of superoxide in RAW 264.7 macrophage cells using microchip electrophoresis coupled to laser-induced fluorescence. Anal Bioanal Chem. 2015;407:7003–12.CrossRefGoogle Scholar
  24. 24.
    Zare RN, Kim S. Microfluidic platforms for single-cell analysis. Annu Rev Biomed Eng. 2010;12:187–201.CrossRefGoogle Scholar
  25. 25.
    Culbertson CT. Single cell analysis on microfluidic devices. Methods Mol Biol. 2006;339:203–16.Google Scholar
  26. 26.
    Price AK, Culbertson CT. Chemical analysis of single mammalian cells with microfluidics. Strategies for culturing, sorting, trapping, and lysing cells and separating their contents on chips. Anal Chem. 2007;79:2614–21.CrossRefGoogle Scholar
  27. 27.
    Lin Y, Trouillon R, Safina G, Ewing AG. Chemical analysis of single cells. Anal Chem. 2011;83:4369–92.CrossRefGoogle Scholar
  28. 28.
    Kojima H, Urano Y, Kikuchi K, Higuchi T, Hirata Y, Nagano T. Fluorescent indicators for imaging nitric oxide production. Angew Chem Int Ed. 1999;38:3209–12.CrossRefGoogle Scholar
  29. 29.
    Kim WS, Ye X, Rubakhin SS, Sweedler JV. Measuring nitric oxide in single neurons by capillary electrophoresis with laser-induced fluorescence: use of ascorbate oxidase in diaminofluorescein measurements. Anal Chem. 2006;78:1859–65.CrossRefGoogle Scholar
  30. 30.
    Balcerczyk A, Soszynski M, Bartosz G. On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide. Free Radic Biol Med. 2005;39:327–35.CrossRefGoogle Scholar
  31. 31.
    Zhang X, Kim WS, Hatcher N, Potgieter K, Moroz LL, Gillette R, et al. Interfering with nitric oxide measurements. 4,5-diaminofluorescein reacts with dehydroascorbic acid and ascorbic acid. J Biol Chem. 2002;277:48472–8.CrossRefGoogle Scholar
  32. 32.
    Fukai T, Ushio-Fukai M. Superoxide dismutases: role in redox signaling, vascular function, and diseases. Antioxid Redox Signal. 2011;15:1583–606.CrossRefGoogle Scholar
  33. 33.
    Vadiveloo PK, Keramidaris E, Morrison WA, Stewart AG. Lipopolysaccharide-induced cell cycle arrest in macrophages occurs independently of nitric oxide synthase II induction. Biochim Biophys Acta. 2001;1539:140–6.CrossRefGoogle Scholar
  34. 34.
    Daigneault M, Preston JA, Marriott HM, Whyte MK, Dockrell DH. The identification of markers of macrophage differentiation in PMA-stimulated THP-1 cells and monocyte-derived macrophages. PLoS One. 2010;5, e8668.CrossRefGoogle Scholar
  35. 35.
    Nishio K, Horie M, Akazawa Y, Shichiri M, Iwahashi H, Hagihara Y, et al. Attenuation of lipopolysaccharide (LPS)-induced cytotoxicity by tocopherols and tocotrienols. Redox Biol. 2013;1:97–103.CrossRefGoogle Scholar
  36. 36.
    Togo T, Katsuse O, Iseki E. Nitric oxide pathways in Alzheimer's disease and other neurodegenerative dementias. Neurol Res. 2004;26:563–6.CrossRefGoogle Scholar
  37. 37.
    Wu Z, Zhao Y, Zhao B. Superoxide anion, uncoupling proteins and Alzheimer's disease. J Clin Biochem Nutr. 2010;46:187–94.CrossRefGoogle Scholar
  38. 38.
    Xia Y, Roman LJ, Masters BS, Zweier JL. Inducible nitric-oxide synthase generates superoxide from the reductase domain. J Biol Chem. 1998;273:22635–9.CrossRefGoogle Scholar
  39. 39.
    Heinzel B, John M, Klatt P, Bohme E, Mayer B. Ca2+/calmodulin-dependent formation of hydrogen peroxide by brain nitric oxide synthase. Biochem J. 1992;281:627–30.CrossRefGoogle Scholar
  40. 40.
    Pou S, Pou WS, Bredt DS, Snyder SH, Rosen GM. Generation of superoxide by purified brain nitric oxide synthase. J Biol Chem. 1992;267:24173–6.Google Scholar
  41. 41.
    Pou S, Keaton L, Surichamorn W, Rosen GM. Mechanism of superoxide generation by neuronal nitric-oxide synthase. J Biol Chem. 1999;274:9573–80.CrossRefGoogle Scholar
  42. 42.
    Hipkiss AR. Carnosine and its possible roles in nutrition and health. Adv Food Nutr Res. 2009;57:87–154.CrossRefGoogle Scholar
  43. 43.
    Caruso G, Fresta CG, Martinez-Becerra F, Antonio L, Johnson RT, de Campos RPS, Siegel JM, Wijesinghe MB, Lazzarino G, Lunte SM. Carnosine modulates nitric oxide in stimulated RAW 264.7 macrophages. Mol Cell Biochem. 2017. doi: 10.1007/s11010-017-2991-3.
  44. 44.
    Fresta CG, Hogard HL, Caruso G, Costa EEM, Lazzarino G, Lunte SM. Monitoring carnosine uptake by RAW 264.7 macrophage cells using microchip electrophoresis with fluorescence detection. Anal Methods. 2017;9:402–8.CrossRefGoogle Scholar
  45. 45.
    Nicholls DG. Mitochondrial calcium function and dysfunction in the central nervous system. Biochim Biophys Acta. 1787;2009:1416–24.Google Scholar
  46. 46.
    Yan Y, Wei CL, Zhang WR, Cheng HP, Liu J. Cross-talk between calcium and reactive oxygen species signaling. Acta Pharmacol Sin. 2006;27:821–6.CrossRefGoogle Scholar
  47. 47.
    Scully SP, Segel GB, Lichtman MA. Relationship of superoxide production to cytoplasmic free calcium in human monocytes. J Clin Investig. 1986;77:1349–56.CrossRefGoogle Scholar
  48. 48.
    Valentin F, Bueb J, Capdeville-Atkinson C, Tschirhart E. Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells. Cell Calcium. 2001;29:409–15.CrossRefGoogle Scholar
  49. 49.
    Fleisher-Berkovich S, Abramovitch-Dahan C, Ben-Shabat S, Apte R, Beit-Yannai E. Inhibitory effect of carnosine and N-acetyl carnosine on LPS-induced microglial oxidative stress and inflammation. Peptides. 2009;30:1306–12.CrossRefGoogle Scholar
  50. 50.
    Nicoletti VG, Santoro AM, Grasso G, Vagliasindi LI, Giuffrida ML, Cuppari C, et al. Carnosine interaction with nitric oxide and astroglial cell protection. J Neurosci Res. 2007;85:2239–45.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Giuseppe Caruso
    • 1
    • 2
  • Claudia G. Fresta
    • 1
    • 2
  • Joseph M. Siegel
    • 1
    • 3
  • Manjula B. Wijesinghe
    • 1
    • 3
  • Susan M. Lunte
    • 1
    • 2
    • 3
  1. 1.Ralph N. Adams Institute for Bioanalytical ChemistryUniversity of KansasLawrenceUSA
  2. 2.Department of Pharmaceutical ChemistryUniversity of KansasLawrenceUSA
  3. 3.Department of ChemistryUniversity of KansasLawrenceUSA

Personalised recommendations