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Detection of in vitro S-nitrosylated Compounds with Cavity Ring-down Spectroscopy

This study demonstrates the implementation of an instrument capable of detecting and measuring S-nitrosylated compounds in vitro using cavity ring-down spectroscopy. The sensitivity of the instrument allows for metabolic studies of S-nitrosothiols through cell growth medium measurements.

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Detection of in vitro S-nitrosylated Compounds with Cavity Ring-down Spectroscopy

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  1. Detection of in vitro S-nitrosylated Compounds with Cavity Ring-down Spectroscopy Mary L. Rad, Monique M. Mezher and Kevin K. Lehmann University of Virginia, Charlottesville, VA Benjamin M. Gaston Case Western Reserve University, Cleveland, OH

  2. Introduction

  3. http://www.nobelprize.org/nobel_prizes/medicine/laureates/1998/illpres/http://www.nobelprize.org/nobel_prizes/medicine/laureates/1998/illpres/

  4. GSNO Cell Membrane

  5. GSH GSNO Cell Membrane Trans-nitrosylation SNO-Cys Cysteine

  6. Cystic Fibrosis Thick mucus not eliminated by cilia Immature CFTR Chloride Channel

  7. GSNO SNO-Cys Cysteine Functioning CFTR Chloride Channel

  8. GSNO SNO-Cys Cysteine L-leucine

  9. GSNO Cysteine L-leucine

  10. Mass Spectrometry Chemiluminescence

  11. Mass Spectrometry Sample Prep M/Z separation M/Z separation Sample ionization fragmentation detection enzymatic digestion, LC, etc. LOD ~5 ppbV Chemiluminescence Pan et al. Sci. Rep. 5: 8725,2005

  12. Mass Spectrometry Sample Prep M/Z separation M/Z separation Sample ionization fragmentation detection enzymatic digestion, LC, etc. LOD ~5 ppbV Chemiluminescence Visible light emitted GE Nitric Oxide Analyzer Fact Sheet

  13. Methods

  14. 14NO R(13/2)3/2 15NO R(15/2)3/2

  15. 2 1 3

  16. 2 1 3

  17. 2 1 3

  18. 2 Optical cavity pressure ~20 torr 1 3 100 sccm flow rate: residence time ~10 s

  19. Results

  20. Raw data calibration curve. LOD (3s) = 8.23 pmoles Slope = 0.0175(2) area units/pmole 50, 100 and 250 mL injections of 3x serial dilutions of GSNO-15. Data convolved with exponentially modified Gaussian function to suppress noise.

  21. Raw data calibration curve. LOD (3s) = 8.23 pmoles Slope = 0.0175(2) area units/pmole 50, 100 and 250 mL injections of 3x serial dilutions of GSNO-15. Data convolved with exponentially modified Gaussian function to suppress noise. EMG convolved data calibration curve. LOD (3σ) = 0.59 pmoles Slope = 0.0179(3) area units/pmole

  22. Biological Sample Measurement Human airway epithelial cells incubated with added 15N-s-nitro-cystamine (CANO) Two cell growth filters RSNO concentration of 10 ± 1 nM

  23. Exponential decay fits of 15NO released vs incubation time y = y0 + Ae-t/t

  24. Exponential decay fits of 15NO released vs incubation time y = y0 + Ae-t/t

  25. Future Work

  26. Circularly polarized light gives DM = +1transitions

  27. Slope = -0.0090108 Slope = -0.010225

  28. Slope = -0.0090108 Slope = -0.010225

  29. Slope = -0.0090108 Slope = -0.010225

  30. Slope = -0.0090108 Slope = -0.010225 180 MHz p-p Laser Frequency Modulation No Laser Frequency Modulation

  31. Conclusions

  32. We have implemented an instrument that is capable of reaching the sensitivity necessary for in vitro metabolic studies of s-nitrosothiols by way of cell growth medium measurements. Additional improvements are necessary to determine s-nitrosothiol concentration directly in cultured cells. Zeeman modulation shows better stability than laser modulation indicating we will have better sensitivity with this new system. Optimizing polarization angle yields slight improvement to ring-down time. L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.

  33. We have implemented an instrument that is capable of reaching the sensitivity necessary for in vitro metabolic studies of s-nitrosothiols by way of cell growth medium measurements. Additional improvements are necessary to determine s-nitrosothiol concentration directly in cultured cells. Zeeman modulation shows better stability than laser modulation indicating we will have better sensitivity with this new system. Optimizing polarization angle yields slight improvement to ring-down time. L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells. Slope = -0.010225

  34. We have implemented an instrument that is capable of reaching the sensitivity necessary for in vitro metabolic studies of s-nitrosothiols by way of cell growth medium measurements. Additional improvements are necessary to determine s-nitrosothiol concentration directly in cultured cells. Zeeman modulation shows better stability than laser modulation indicating we will have better sensitivity with this new system. Optimizing polarization angle yields slight improvement to ring-down time. L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.

  35. We have implemented an instrument that is capable of reaching the sensitivity necessary for in vitro metabolic studies of s-nitrosothiols by way of cell growth medium measurements. Additional improvements are necessary to determine s-nitrosothiol concentration directly in cultured cells. Zeeman modulation shows better stability than laser modulation indicating we will have better sensitivity with this new system. Optimizing polarization angle yields slight improvement to ring-down time. L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.

  36. Acknowledgements I would like to acknowledge the following for their help and support in the proposal of this project: Laura Smith, VitaliStsiapura, Gerard Wysocki Financial support: UVA, NSF, and NIH

  37. Questions?

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