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. Nitric oxide has been of biological interest for nearly 40 years due to its role in cardiovascular and nervous signaling. In particular, three doctors were awarded the Nobel Prize in 1998 for the discovery of the role of nitric oxide in the relaxation of cardiovascular smooth muscle.

4. GSNO
Cell Membrane
As nitric oxide is a radical, donor molecules are necessary to transport NO safely throughout the body so that it can be applied biochemically where appropriate.
It has been shown that s-nitrosothiols (most notably s-nitrosoglutathione or GSNO) are important NO carrier molecules. In particular GSNO is a known therapeutic agents in Alzheimer's Disease, ovarian cancer, asthma, and cystic fibrosis.

5. GSH GSNO Cell Membrane Trans-nitrosylation SNO-Cys Cysteine
Chemicals must reach the inside of the cell in order to act therapeutically. Studies have shown that GSNO is unable to cross the cell membrane on its own. However, it has been shown that by undergoing a trans-nitrosylation reaction with cysteine to form s-nitrosocysteine the NO can be transferred to a molecule which can cross the cell membrane.

6. Cystic Fibrosis Thick mucus not eliminated by cilia
In patients with cystic fibrosis, mutations causing decreased maturation, or premature destruction, of the cystic fibrosis trans-membrane conductance regulator chloride channel impede movement of salt and water across the cell membrane. This causes the thick mucus lining the lungs and other organs that is characteristic of this disease.
Immature CFTR Chloride Channel

7. Functioning CFTR Chloride Channel
GSNO
SNO-Cys
Cysteine
It was shown that GSNO increased maturation of the cystic fibrosis trans-membrane conductance regulator chloride channel making it a potential therapeutic target for cystic fibrosis and other diseases with similar CFTR mutations.
Functioning CFTR Chloride Channel

8. GSNO Cysteine SNO-Cys L-leucine
To understand how GSNO is acting in the human body studies were performed with the addition of the amino acid L-Leucine. It was found that in the presence of l-leucine membrane transport of RSNOs is prevented…
L-leucine

9. GSNO Cysteine L-leucine
…and so is the enhancement in maturation of CFTR protein causing chloride ions to become trapped inside the cell.
L-leucine

10. Mass Spectrometry Chemiluminescence
Two popular ways to do perform NO detection from biological samples are Mass Spectrometry and Chemiluminescence.

11. Mass Spectrometry Chemiluminescence LOD ~5 ppbV Sample Prep
ionization
M/Z separation
Sample
fragmentation
detection
Sample Prep
enzymatic digestion, LC, etc.
LOD ~5 ppbV
Chemiluminescence
While mass spectrometry is extremely sensitive, it requires considerable sample preparation even before the ionization stage of the experiment. This sample preparation includes processing such as enzymatic digestion of proteins and liquid chromatography, both of which can break or make S-NO bonds resulting in an incorrect final RSNO concentration measurement.
Pan et al. Sci. Rep. 5: 8725, 2005

12. Mass Spectrometry Chemiluminescence LOD ~5 ppbV Visible light emitted
ionization
M/Z separation
Sample
fragmentation
detection
Sample Prep
enzymatic digestion, LC, etc.
LOD ~5 ppbV
Chemiluminescence
Chemiluminescence, while being a sensitive and relatively simple technique, has no isotopic specificity and is therefore not possible to determine endogenous vs exogenous source of NO. This makes metabolic studies driven by isotopic labeling used for screening therapeutic targets impossible.
Visible light emitted
GE Nitric Oxide Analyzer Fact Sheet

13. Methods

14. To study RSNO concentration in biological samples we built a cavity ring-down spectrometer centered around 5.3 microns to measure the amount of NO and 15NO released by these samples.

15. The light is generated by a Daylight Solutions EC-QCL
The light is generated by a Daylight Solutions EC-QCL. The IR then runs through an optical isolator and into an acousto-optic modulator where the first order beam is sent through mode matching optics and into the optical cavity. The zero order beam is omitted here for clarity.

16. When a specified trigger threshold is reached the AOM is switched off using a digital delay generator, extinguishing the first order beam. Ring-down is then acquired with a TiePie Handyscope and analyzed using a custom program written in Delphi.

17. 14NO
R(13/2)3/2
15NO
R(15/2)3/2
On the left is the HITRAN simulation of 14 and 15NO, water, and carbon dioxide in the spectral region available with our laser. On the right are shown the spectral line simulations of 14NO R(13/2)3/2 and 15NO R(15/2)3/2

18. 2 1 3
To measure the biological samples we have three possible reaction vessels. Two vessels rely on injection of the sample and one allows us to measure directly from the microtubes the samples are transported in.

19. 2 1 3
To begin, samples are injected to the reaction vessel, or are preloaded in the case of vessel 2.

20. 2 1 3
Copper cysteine is injected to break the S-N bond in the RSNO to release the NO to be measured. In the case of reaction vessel 3 UV light is also used to further insure complete liberation of NO.

21. Optical cavity pressure ~20 torr1 3
Helium carrier gas is bubbled through the sample, and the NO and helium run through a dry ice and acetone cold trap and into the optical cavity for NO absorption measurements.
100 sccm flow rate: residence time ~10 s

22. Results

23. Standard injection of 15N-GSNO

24. Raw data calibration curve.
LOD (3s) = 8.23 pmoles
Slope = (2) area units/pmole
Initial calibration data indicated our 15NO three sigma limit of detection was 8.23 pmoles. It was believed that we could increase sensitivity in post processing by convolving the data with an exponentially modified Gaussian curve.
50, 100 and 250 mL injections of 3x serial dilutions of GSNO-15. Data convolved with exponentially modified Gaussian function to suppress noise.

26. Raw data calibration curve.
LOD (3s) = 8.23 pmoles
Slope = (2) area units/pmole
We found that through this convolution we were able to reduce the three sigma limit of detection to 0.59 pmoles. You can see the three standard injection times marked by a white line. In the green, unfiltered data it is impossible to see the NO released from any but the most concentrated injection. However with filtering we are able to see peaks from all three injections.
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 (3s) = 0.59 pmoles
Slope = (3) area units/pmole

27. 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
N-s-nitrosystamine (CANO) is a potential therapeutic target for cystic fibrosis due to its similarity to GSNO. 15N-s-nitrocystamine was incubated with human airway epithelial cells to determine if it could cross the cell membrane. Only once were we able to measure NO released directly from cell growth filters and this was with two filters at high CANO incubation concentration. Future studies focused on measuring CANO reduction from the surrounding cell growth medium to determine if CANO uptake was occurring.

28. 1/t (s-1) -leucine +leucine control 0.01 ±0.5
Control trials were performed where seven samples of cell growth medium with CANO without cells present to determine the concentration over time in the absence of biological processing. This data was fit with an exponential decay consistent with first-order kinetics typically seen in biochemical reactions.
1/t (s-1)
-leucine
+leucine
control
0.01 ±0.5
Exponential decay fits of 15NO released vs incubation time y = y0 + Ae-t/t

29. 1/t (s-1) -leucine 0.1 ±0.06 +leucine 0.1 ±0.2 control 0.01 ±0.5
Addition of leucine showed an overall decrease in concentration of CANO in the cell growth medium, however the first-order rate constant remained the same with and without leucine. If leucine were acting on CANO uptake in a similar way to GSNO we would expect to see more CANO and a slower rate in the +leucine cell growth medium than then –leucine cell growth medium. This could indicate CANO crosses the cell membrane using a different mechanism than GSNO that is not affected by leucine.
1/t (s-1)
-leucine
0.1 ±0.06
+leucine
0.1 ±0.2
control
0.01 ±0.5
Exponential decay fits of 15NO released vs incubation time y = y0 + Ae-t/t

30. Future Work

31. Through Zeeman modulation, we are able to achieve a maximum splitting of about 10 MHz, however if we were using the linearly polarized light generated by our QCL this would only result in a broadening of the NO absorption.

32. Circularly polarized light gives DM = +1 transitions
Due to selection rules, we are mostly observing the transition into the highest m level, so we are getting the most modulation of the NO absorption possible, which should give us the best discriminator curve for laser locking.

33. To lock the laser onto the center of the NO absorption, rather than modulating the laser frequency, we modulate the absorption frequency in an NO reference cell using Zeeman modulation. A solenoid is driven by a function generator amplified by an audio amplifier to achieve an oscillating magnetic field of gauss. By using a quarter wave plate before the reference cell we are able to convert the linearly polarized light from the QCL to circularly polarized light.

34. Additionally, we actively correct the optical cavity length using three PZTs so that the center of the cavity mode is at the center of the NO absorption. In this way we are able to maximize sensitivity to NO released by maintaining our frequency at the line center and frequency stability of the laser insuring optimum coupling into the optical cavity.

35. Slope =
Slope =
We wanted to compare laser locking with laser modulation and Zeeman modulation. Here I am showing 1kHz laser current modulation on the left and 4.4 kHz Zeeman modulation on the right.
Using Zeeman modulation gives similar corrective ability with less noise to the laser locking as the traditional laser current modulation. Zeeman modulation has the benefit of not changing the laser frequency which allows the absorption to remain on the line center for the entirety of the experiment and allows for better coupling into the ring-down cavity. This results in more stability and sensitivity for NO detection.

36. Slope =
Slope =
Using Zeeman modulation gives similar corrective ability…

37. Slope =
Slope =
… with less noise in the discriminator signal as the traditional laser current modulation.
Zeeman modulation has the benefit of not changing the laser frequency which allows the absorption to remain on the line center for the entirety of the experiment and allows for better coupling into the ring-down cavity. This results in more stability and sensitivity for NO detection.

38. 180 MHz p-p Laser Frequency Modulation No Laser Frequency Modulation
Slope =
Slope =
Additionally, Zeeman modulation has the benefit of not changing the laser frequency which allows the absorption to remain on the line center for the entirety of the experiment. Thisl allows for better coupling into the ring-down cavity and more stability and sensitivity for NO detection.
180 MHz p-p Laser Frequency Modulation
No Laser Frequency Modulation

39. Conclusions

40. 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.
L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.
Images for this slide

41. 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.
L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.
Slope =
Images for this slide

42. 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.
L-leucine has undetermined effect on CANO uptake, but control trials show CANO is being broken down by human airway epithelial cells.
Images for this slide

43. Acknowledgements
I would like to acknowledge the following for their help and support in the proposal of this project:
Laura Smith, Vitali Stsiapura, Gerard Wysocki
Financial support: UVA, NSF, and NIH
What should I put on this slide?

44. Questions?