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

5. NO Measurement Techniques 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 NO 2 TDLAS Molecules absorb IR light Simultaneous NO and CO 2 detection improves reliability Patients can exhale at different flow rates No consumables NO + O 3  NO 2 + h (light) H 2 O+CO 2 CO 2 NO Vibrational Frequency Spectral Signals

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

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

8. Treatment Monitoring

9. 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. Typical eNO Range (5-70 ppb) Individual Vehicles Group of Vehicles Stopped at Intersection High Emission Vehicles Street Level NO (Norman, OK) Important question for polluted areas

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

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

12. Biochemistry of NO Absorption Ambient NO is rapidly absorbed by the airways and not subsequently exhaled. 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. O 2, NO, etc. CO 2, etc. NO + ROS  NO 2 – and NO 3 – Airway Tissue

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.