Annals of Biomedical Engineering

, Volume 41, Issue 4, pp 827–836 | Cite as

Surface Fluorescence Studies of Tissue Mitochondrial Redox State in Isolated Perfused Rat Lungs

  • Kevin Staniszewski
  • Said H. Audi
  • Reyhaneh Sepehr
  • Elizabeth R. Jacobs
  • Mahsa RanjiEmail author


We designed a fiber-optic-based optoelectronic fluorometer to measure emitted fluorescence from the auto-fluorescent electron carriers NADH and FAD of the mitochondrial electron transport chain (ETC). The ratio of NADH to FAD is called the redox ratio (RR = NADH/FAD) and is an indicator of the oxidoreductive state of tissue. We evaluated the fluorometer by measuring the fluorescence intensities of NADH and FAD at the surface of isolated, perfused rat lungs. Alterations of lung mitochondrial metabolic state were achieved by the addition of rotenone (complex I inhibitor), potassium cyanide (KCN, complex IV inhibitor) and/or pentachlorophenol (PCP, uncoupler) into the perfusate recirculating through the lung. Rotenone- or KCN-containing perfusate increased RR by 21 and 30%, respectively. In contrast, PCP-containing perfusate decreased RR by 27%. These changes are consistent with the established effects of rotenone, KCN, and PCP on the redox status of the ETC. Addition of blood to perfusate quenched NADH and FAD signal, but had no effect on RR. This study demonstrates the capacity of fluorometry to detect a change in mitochondrial redox state in isolated perfused lungs, and suggests the potential of fluorometry for use in in vivo experiments to extract a sensitive measure of lung tissue health in real-time.


Lung surface fluorometry Nicotinamide Adenine Dinucleotide (NADH) Flavin Adenine Dinucleotide (FADH2) Mitochondrial redox 



We appreciate the support of University of Wisconsin Milwaukee RGI 6 Grant, Clinical and Translational Science Institute KL2 Grant: NIH 8Kl2TR000056, Wisconsin Applied Research grant (Wi-ARG), NIH Grants HL-24349 (SHA), HL 49294 (ERJ), and the Department of Veterans Affairs that provided resources essential to the completion of these investigations. We acknowledge the technical help from Dr. Steven Haworth in the Clement J. Zablocki VA, Milwaukee, WI.


  1. 1.
    Aldakkak, M., et al. Modulation of mitochondrial bioenergetics in the isolated Guinea pig beating heart by potassium and lidocaine cardioplegia: implications for cardioprotection. J. Cardiovasc. Pharmacol. 54:298–309, 2009.PubMedCrossRefGoogle Scholar
  2. 2.
    Aldrich, T. K., et al. Paraquat inhibits mixed-function oxidation by rat lung. J. Appl. Physiol. 54:1089–1093, 1983.PubMedGoogle Scholar
  3. 3.
    Allen, C. B., and C. W. White. Glucose modulates cell death due to normobaric hyperoxia by maintaining cellular ATP. Am. J. Physiol. 274:L159–L164, 1998.PubMedGoogle Scholar
  4. 4.
    Audi, S. H., et al. Duroquinone reduction during passage through the pulmonary circulation. Am. J. Physiol. Lung Cell. Mol. Physiol. 285:L1116–L1131, 2003.PubMedGoogle Scholar
  5. 5.
    Audi, S. H., et al. Coenzyme Q1 redox metabolism during passage through the rat pulmonary circulation and the effect of hyperoxia. J. Appl. Physiol. 105:1114–1126, 2008.PubMedCrossRefGoogle Scholar
  6. 6.
    Balaban, R. S., and L. J. Mandel. Coupling of aerobic metabolism to active ion transport in the kidney. J. Physiol. 304:331–348, 1980.PubMedGoogle Scholar
  7. 7.
    Barlow, C. H., et al. Fluorescence mapping of mitochondrial redox changes in heart and brain. Crit. Care Med. 7:402–406, 1979.PubMedCrossRefGoogle Scholar
  8. 8.
    Boldt, M., et al. A sensitive dual wavelength microspectrophotometer for the measurement of tissue fluorescence and reflectance. Pflugers Arch. 385:167–173, 1980.PubMedCrossRefGoogle Scholar
  9. 9.
    Brandes, R., and D. M. Bers. Increased work in cardiac trabeculae causes decreased mitochondrial NADH fluorescence followed by slow recovery. Biophys. J. 71:1024–1035, 1996.PubMedCrossRefGoogle Scholar
  10. 10.
    Chance, B., and H. Baltscheffsky. Respiratory enzymes in oxidative phosphorylation. VII. Binding of intramitochondrial reduced pyridine nucleotide. J. Biol. Chem. 233:736–739, 1958.PubMedGoogle Scholar
  11. 11.
    Chance, B., et al. Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals. J. Biol. Chem. 254:4764–4771, 1979.PubMedGoogle Scholar
  12. 12.
    Commoner, B., and D. Lipkin. The application of the Beer-Lambert law to optically anisotropic systems. Science 110:41–43, 1949.PubMedCrossRefGoogle Scholar
  13. 13.
    Crapo, J. D., et al. Structural and biochemical changes in rat lungs occurring during exposures to lethal and adaptive doses of oxygen. Am. Rev. Respir. Dis. 122:123–143, 1980.PubMedGoogle Scholar
  14. 14.
    De Blasi, R. A., et al. Noninvasive measurement of forearm blood flow and oxygen consumption by near-infrared spectroscopy. J. Appl. Physiol. 76:1388–1393, 1994.PubMedGoogle Scholar
  15. 15.
    Else, P. L., and A. J. Hulbert. Mammals: an allometric study of metabolism at tissue and mitochondrial level. Am. J. Physiol. 248:R415–R421, 1985.PubMedGoogle Scholar
  16. 16.
    Eng, J., et al. Nicotinamide adenine dinucleotide fluorescence spectroscopy and imaging of isolated cardiac myocytes. Biophys. J. 55:621–630, 1989.PubMedCrossRefGoogle Scholar
  17. 17.
    Fisher, A. B. Intermediary metabolism of the lung. Environ. Health Perspect. 55:149–158, 1984.PubMedCrossRefGoogle Scholar
  18. 18.
    Fisher, A. B., et al. Evaluation of redox state of isolated perfused rat lung. Am. J. Physiol. 230:1198–1204, 1976.PubMedGoogle Scholar
  19. 19.
    Fisher, A. B., et al. Pulmonary mixed-function oxidation: stimulation by glucose and the effects of metabolic inhibitors. Biochem. Pharmacol. 30:379–383, 1981.PubMedCrossRefGoogle Scholar
  20. 20.
    Gan, Z., et al. Quantifying mitochondrial and plasma membrane potentials in intact pulmonary arterial endothelial cells based on extracellular disposition of rhodamine dyes. Am. J. Physiol. Lung Cell. Mol. Physiol. 300:L762–L772, 2011.PubMedCrossRefGoogle Scholar
  21. 21.
    Gerich, F. J., et al. Mitochondrial inhibition prior to oxygen-withdrawal facilitates the occurrence of hypoxia-induced spreading depression in rat hippocampal slices. J. Neurophysiol. 96:492–504, 2006.PubMedCrossRefGoogle Scholar
  22. 22.
    Kunz, W. S., and F. N. Gellerich. Quantification of the content of fluorescent flavoproteins in mitochondria from liver, kidney cortex, skeletal muscle, and brain. Biochem. Med. Metab. Biol. 50:103–110, 1993.PubMedCrossRefGoogle Scholar
  23. 23.
    Kunz, W. S., and W. Kunz. Contribution of different enzymes to flavoprotein fluorescence of isolated rat liver mitochondria. Biochim. Biophys. Acta 841:237–246, 1985.PubMedCrossRefGoogle Scholar
  24. 24.
    Liu, Q., et al. Investigation of synchronous fluorescence method in multicomponent analysis in tissue. IEEE J. Sel. Top. Quantum Electron. 16:14, 2010.Google Scholar
  25. 25.
    Maleki, S., et al. Mitochondrial redox studies of oxidative stress in kidneys from diabetic mice. Biomed. Opt. Express 3:273–281, 2012.PubMedCrossRefGoogle Scholar
  26. 26.
    Matsubara, M., et al. In vivo fluorometric assessment of cyclosporine on mitochondrial function during myocardial ischemia and reperfusion. Ann. Thorac. Surg. 89:1532–1537, 2010.PubMedCrossRefGoogle Scholar
  27. 27.
    Mayevsky, A. Brain NADH redox state monitored in vivo by fiber optic surface fluorometry. Brain Res. 319:49–68, 1984.PubMedGoogle Scholar
  28. 28.
    Meirovithz, E., et al. Effect of hyperbaric oxygenation on brain hemodynamics, hemoglobin oxygenation and mitochondrial NADH. Brain Res. Rev. 54:294–304, 2007.PubMedCrossRefGoogle Scholar
  29. 29.
    Mycek, M. A., et al. Colonic polyp differentiation using time-resolved autofluorescence spectroscopy. Gastrointest. Endosc. 48:390–394, 1998.PubMedCrossRefGoogle Scholar
  30. 30.
    Nioka, S., et al. Simulation of Mb/Hb in NIRS and oxygen gradient in the human and canine skeletal muscles using H-NMR and NIRS. Adv. Exp. Med. Biol. 578:223–228, 2006.PubMedCrossRefGoogle Scholar
  31. 31.
    Nuutinen, E. M. Subcellular origin of the surface fluorescence of reduced nicotinamide nucleotides in the isolated perfused rat heart. Basic Res. Cardiol. 79:49–58, 1984.PubMedCrossRefGoogle Scholar
  32. 32.
    Owen, M. R., et al. Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain. Biochem. J. 348(Pt 3):607–614, 2000.PubMedCrossRefGoogle Scholar
  33. 33.
    Ramanujam, N., et al. In vivo diagnosis of cervical intraepithelial neoplasia using 337-nm-excited laser-induced fluorescence. Proc. Natl Acad. Sci. USA 91:10193–10197, 1994.PubMedCrossRefGoogle Scholar
  34. 34.
    Ramanujam, N., et al. Low temperature fluorescence imaging of freeze-trapped human cervical tissues. Opt. Express 8:335–343, 2001.PubMedCrossRefGoogle Scholar
  35. 35.
    Ranji, M. Fluorescent images of mitochondrial redox states of in situ mouse hypoxic ischemic intestines. J. Innov. Opt. Health Sci. (JIOHS) 2:365–374, 2009.CrossRefGoogle Scholar
  36. 36.
    Ranji, M., et al. Fluorescence spectroscopy and imaging of myocardial apoptosis. J. Biomed. Opt. 11:064036, 2006.PubMedCrossRefGoogle Scholar
  37. 37.
    Ranji, M., et al. Quantifying acute myocardial injury using ratiometric fluorometry. IEEE Trans. Biomed. Eng. 56:1556–1563, 2009.PubMedCrossRefGoogle Scholar
  38. 38.
    Rocheleau, J. V., et al. Quantitative NAD(P)H/flavoprotein autofluorescence imaging reveals metabolic mechanisms of pancreatic islet pyruvate response. J. Biol. Chem. 279:31780–31787, 2004.PubMedCrossRefGoogle Scholar
  39. 39.
    Sepehr, R., et al. Optical imaging of tissue mitochondrial redox state in intact rat lungs in two models of pulmonary oxidative stress. J. Biomed. Opt. 17:046010, 2012.PubMedCrossRefGoogle Scholar
  40. 40.
    Vollmar, B., et al. A correlation of intravital microscopically assessed NADH fluorescence, tissue oxygenation, and organ function during shock and resuscitation of the rat liver. Adv. Exp. Med. Biol. 454:95–101, 1998.PubMedCrossRefGoogle Scholar
  41. 41.
    Xia, L., et al. Reduction of ubiquinone by lipoamide dehydrogenase. An antioxidant regenerating pathway. Eur. J. Biochem. 268:1486–1490, 2001.PubMedCrossRefGoogle Scholar
  42. 42.
    Zmijewski, J. W., et al. Mitochondrial respiratory complex I regulates neutrophil activation and severity of lung injury. Am. J. Respir. Crit. Care Med. 178:168–179, 2008.PubMedCrossRefGoogle Scholar

Copyright information

© Biomedical Engineering Society 2012

Authors and Affiliations

  • Kevin Staniszewski
    • 1
  • Said H. Audi
    • 2
  • Reyhaneh Sepehr
    • 1
  • Elizabeth R. Jacobs
    • 3
    • 4
  • Mahsa Ranji
    • 1
    Email author
  1. 1.Biophotonics Lab, Department of Electrical EngineeringUniversity of Wisconsin MilwaukeeMilwaukeeUSA
  2. 2.Department of Biomedical EngineeringMarquette UniversityMilwaukeeUSA
  3. 3.Research and DevelopmentClement J. Zablocki VA Medical CenterMilwaukeeUSA
  4. 4.Medical College of WisconsinMilwaukeeUSA

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