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 TDLASInsensitivity to High Atmospheric

Nitric Oxide Concentrations

ACAAI Annual Meeting San Antonio, TexasNovember 18, 2002

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.

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.

Excessive NO is associated with airway inflammation.Excessive NO is associated with airway inflammation.

Nitric Oxide Biochemistry

Effects of NOVasodilatationHost DefenseSmooth Muscle Relaxation

Healthy SubjectscNOS + L-arginine L-citrulline + NO

AsthmaticscNOS + L-arginine L-citrulline + NO iNOS + L-arginine L-citrulline + NO

iNOS Expression Caused by epithelial cell exposure to specific cytokines and endotoxins

Epithelial CellEpithelial 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

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 NO2

TDLAS• Molecules absorb IR light

• Simultaneous NO and CO2 detection improves reliability

• Patients can exhale at different flow rates

• No consumables

1Laser Chip Detector

1Laser Chip DetectorLaser Chip Detector

2Laser Chip Detector

2Laser Chip Detector

NO + O3 NO2 + h (light)

Wavenumber (cm-1)

1912.7 1912.8 1912.9 1913.0

Intensity

(cm-1

/molecule x cm

-2

)

10-2710-2610-2510-2410-2310-2210-2110-20

Voltage (V)

-0.090

-0.045

0.000

0.045

0.090

Voltage (V)

-4.0

0.0

4.0

8.0

12.0

CO2

CO2

NO

CO2 + H2O

H2O - Spectral Reference

ETALON

HITRAN

H2O+CO2 CO2

NOVibrational Frequency

Spectral SignalsSpectral Signals

eNO Measurements with TDLAS

Single Exhalation Against Resistance (10-20 sec)

Results in 20 seconds

eNOeCO2

eNOeNOeCO2eCO2

Upper Airway or Nasal NO (nNO)

Lower Airway NO (eNO)

40

20

0eNO

Co

nce

ntr

atio

n (

pp

b)

0

2

4

6

0 5 25 45Time (seconds)

0 5 25 45Time (seconds)Start Exhalation End Exhalation

eCO

2C

on

cen

tra

tio

n (

%)

End Tidal CO2

Laser Light Path(~ 5.2 m)

Breath FlowBreath Flow

ExhaledBreath

Flowing throughGas Cell

ExhaledBreath

Flowing throughGas Cell

Laser and Detector Assembly

Laser and Detector Assembly

Time (seconds)

0 5 10 15 20 25 30 35

eNO

(pp

b)

0

4

8

12

16

20

24

eCO

2 (%

)

0

1

2

3

4eNO = 8.9 ppb

NO

CO2

Asthmatic Examples

Inflamed Airway

Age 14, Male, Mild-PersistentNon-Treated

Normal Airway

Age 5, Female, Mild-PersistentCorticosteriod Treated

Time (seconds)

0 10 20 30 40

eN

O (

pp

b)

0

10

20

30

40

50

60

eC

O2

(%)

0

2

4

6

8

10

12eNO = 52.9 ppb

NO

CO2

Treatment Monitoring

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.

Time of Day

3:55 PM 4:00 PM 4:05 PM 4:10 PM 4:15 PM

Nitr

ic O

xide

Con

cent

ratio

n (p

pb)

0

100

200

300

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

Time (seconds)

0 5 10 15 20 25 30 35 40 45

eN

O (

ppb

)

0

20

40

60

80

eC

O2

(%)

0

2

4

6

8

10

12

14

aNO = 2 ppb

eNO = 74 ppb NO

CO2

Time (seconds)

0 5 10 15 20 25 30 35 40 45

eN

O (

ppb

)

0

20

40

60

80

eC

O2

(%)

0

2

4

6

8

10

12

14eNO = 64 ppb

aNO = 24 ppb

NO

CO2

Time (seconds)

0 5 10 15 20 25 30 35 40 45

eNO

(p

pb)

0

10

20

30

eCO

2 (%

)

0

1

2

3

4eNO = 4 ppb

aNO ~ 2 ppb

CO2

NO

Time (seconds)

0 5 10 15 20 25 30 35 40 45

eNO

(pp

b)

0

10

20

30

eCO

2 (%

)

0

1

2

3

4eNO = 5 ppb CO2

NO

aNO = 22 ppb

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)Low aNO (2 ppb) High aNO (24 ppb)High aNO (24 ppb)

Replication Precision

eNO Range

Time (Days)0 15 30 45 60 75 90

Nitr

ic O

xide

Con

cent

ratio

n (p

pb)

0

10

20

30

40Exhaled NO

Atmospheric NO

AtmosphericNO Range

• 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

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.

O2, NO, etc.

CO2, etc.

NO + ROS NO2

– and NO3

–NO + ROS NO2

– and NO3

–

Airway Tissue

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.