Mark Camp, M.D., F.A.C.P., F.C.C.P. The Lung Center, Norman, OK

1. Exhaled Nitric Oxide Measured using TDLASInsensitivity to High Atmospheric Nitric Oxide Concentrations Mark Camp, M.D., F.A.C.P., F.C.C.P. The Lung Center, Norman, OK James D. Jeffers, Chad Roller, Khosrow Namjou, Ph.D. Ekips Technologies, Inc., Norman, OK Patrick J. McCann, Ph.D. University of Oklahoma, School of Electrical and Computer Engineering ACAAI Annual Meeting San Antonio, Texas November 18, 2002

2. Study Objectives • Evaluate Tunable Diode Laser Absorption Spectroscopy (TDLAS) as a new tool for exhaled nitric oxide (eNO) measurements. • Use eNO to assess airway inflammation and monitor therapy. • Determine if high levels of ambient NO affect eNO measurements.

3. Motivation • “Asthma is a chronic inflammatory disorder of the lower airways…” • Working definition (NHLBI, 1995) • High eNO levels are linked to inflammation. • Spirometry provides only limited and indirect information regarding airway inflammation.

4. Nitric Oxide Biochemistry Healthy Subjects cNOS + L-arginine  L-citrulline + NO Effects of NO Vasodilatation Host Defense Smooth Muscle Relaxation Asthmatics cNOS + L-arginine  L-citrulline + NO iNOS + L-arginine  L-citrulline + NO Excessive NO is associated with airway inflammation. Constitutive and inducible nitric oxide synthases (cNOS and iNOS) are genetically-produced enzymes • iNOS Inhibitors • Inhaled corticosteroids iNOS Expression Caused by epithelial cell exposure to specific cytokines and endotoxins Epithelial Cell  Inflammation  High exhaled NO • Inflammation • Lower eNO

5. Chemiluminescence Reaction with ozone Patients must exhale at constant flow rates Requires NO-free air to zero the instrument Requires calibration using standard reference gases Unreliable calibration gases can result in unreliable data. NO degrades to NO2 TDLAS Molecules absorb IR light Simultaneous NO and CO2 detection improves reliability Patients can exhale at different flow rates No consumables H2O+CO2 Vibrational Frequency CO2 NO NO Measurement Techniques NO + O3 NO2 + h (light) Spectral Signals

6. Breath Flow Laser Light Path (l ~ 5.2 mm) Laser and Detector Assembly Exhaled Breath Flowing through Gas Cell Single Exhalation Against Resistance (10-20 sec) eNO Measurements with TDLAS Results in 20 seconds

7. Asthmatic Examples Age 5, Female, Mild-Persistent Corticosteriod Treated Age 14,Male, Mild-Persistent Non-Treated Normal Airway Inflamed Airway

8. Treatment Monitoring

9. Important question for polluted areas Group of Vehicles Stopped at Intersection Street Level NO (Norman, OK) High Emission Vehicles Individual Vehicles Typical eNO Range (5-70 ppb) Air Pollution Effects? • Nitric Oxide is a major component of smog. • Automobile exhaust is primary source. • Ambient NO (aNO) concentrations will vary with geography, weather conditions (inversion layers), and time of day.

10. eNO not Affected by aNO Low aNO (2 ppb) High aNO (24 ppb) Untreated Asthmatic Untreated Asthmatic Non- Asthmatic Non- Asthmatic Ambient Air Displaced by Breath

11. Replication Precision • TDLAS provides good replication precision in spite of varying atmospheric NO levels. Non-asthmatic volunteer over a three-month period

12. O2, NO, etc. CO2, etc. Biochemistry of NO Absorption • Ambient NO is rapidly absorbed by the airways and not subsequently exhaled. Airway Tissue NO + ROS  NO2– and NO3– • Endogenously-produced NO scavenges cytotoxic reactive oxidative species (ROS). Dweik et al., Proc. Natl. Acad. Sci.98, 2622 (2001) • High eNO is possibly associated with a protective mechanism in asthmatic airways.

13. Conclusions and Future Work • TDLAS successfully measured high eNO in untreated asthmatics and low eNO in treated asthmatics and non-asthmatics. • A reduction in eNO from more than 40 ppb to less than 20 ppb over a 7-day period was observed following anti-inflammatory treatment. • High levels of ambient NO are rapidly absorbed and not subsequently exhaled. • Clinical trials are continuing.