A method for off-line nasal nitric oxide measurement. (Original Article).

Abstract

We conducted a study to test the accuracy of an immediate and delayed off-line technique for measuring nasal nitric oxide (NO) by comparing it with on-line measurements. With the assistance of one volunteer, we obtained these measurements during 30 sessions over a period of 2 months. Off-line measurements were made immediately following the acquisition of NO samples and 1, 2, and 4 hours later. NO samples were obtained from nasal air collected in syringes. We found that the correlation between on-line measurements and the immediate and delayed off-line measurements ranged from 95 to 98%, according to a Bland-Altman analysis. We conclude that off-line nasal NO measurements can be reliably used in clinical practice and research projects, thus obviating the need for patients/subjects to be in close proximity to the analyzer. Off-line nasal NO measurements can effectively substitute for on-line measurements when the latter technique is not practical.

Introduction

The presence of endogenous nitric oxide (NO) in the exhaled breath of humans and animals was first reported by Gustafsson et al in 1991. (1) Since then, interest in this topic has increased. It is now known that exhaled NO originates primarily in the upper airways; the lower airways and lungs make only a minor contribution. (2) NO has several functions. It plays a role in immunity and in the host defense of the upper respiratory system, (3-5) it is a regulator of ciliary motility in the human sinonasal mucosa, (6,7) and it is a modulator of pulmonary function. (8,9) NO is also a marker of inflammation. (2)

The amount of nasal NO output can be altered by some respiratory diseases. For example, output is increased in the presence of allergic rhinitis (4,10) and decreased in cystic fibrosis, (11) Kartagener's syndrome, (12) and sinusitis. (13,14) Therefore, measurement of nasal NO can serve as a noninvasive tool in the diagnosis of sinonasal diseases.

The on-line measurement of NO requires several pieces of bulky hardware and a dedicated station. To obtain maximum advantage of on-line NO measurements in the clinical setting, the equipment and all its components must be located either in the clinic, which is usually busy and crowded, or in a remote laboratory, which requires that the patient be transferred. The alternative is to collect a sample in the clinic and transfer it to the central laboratory for off-line measurement.

In an effort to refine the latter option, we developed a nasal NO collection and transport system that employs special syringes. Our technique was inspired by those that are used for lower-airway sampling (15,16) and by personal communication (June 2000) with P.G. Djupesland, MD, and A. Qian, MD, following the publication of their abstract. (17) Several articles have been written about online NO measurement techniques, (3,15,16,18-21) but published data on off-line methods are limited. The purpose of this article is to describe our validation of an off-line nasal NO measurement technique.

Materials and methods

One of the authors (C.V.)--a 41-year-old male nonsmoker with mild allergic rhinitis--volunteered to act as the subject of this study. Nasal examination revealed that the subject had a mild septal deviation that obstructed less than 25% of the left nasal passage. Skin-prick testing was positive for dust mites (Dermatophagoides pteronyssinus) and seasonal allergens (Kentucky bluegrass, orchard grass, and redtop grass). Because the subject's symptoms were mild, he took no medication for his allergic rhinitis during the study period. The subject also experienced a viral infection of the upper respiratory tract during the study, which increased the amount of nasal NO in his exhaled breath.

NO was measured with a rapid-response chemiluminescent analyzer (NOA 280; Sievers Instruments; Boulder, Cob.). The sampling flow of the analyzer pump was 0.2 L/min. A 2-point calibration was performed daily, first to 0 with air passed through an NO scrubber tube containing KMn[O.sub.4] (potassium permanganate) and activated charcoal, and then with certified standardized NO gas (45 parts per million) for the span (Datex-Ohmeda; Madison, Wis.). The amount of NO in ambient air (baseline) was recorded before each on-line measurement.

The NO analyzer signal output was fed into a computer data acquisition program (NO analysis software for NOA 280, Version 3.00 PNE; Sievers Instruments). The program featured a real-time display of NO vs time written directly into the computer's hard disk as a data file. This program plotted NO concentrations against time and printed the results in graph form.

During each measurement, on- and off-line NO samples were taken at a constant flow of 5 L/min. Flow was created by a suction pump, and it was continuously monitored by a highly accurate flow meter (Aalborg Instruments; Monsey, N.Y.). Baseline NO concentrations were subtracted from measured concentrations before any data were analyzed.

A latex nasal olive (ENTsol Adapter; Kenwood Therapeutics; Fairfield, N.J.) was gently introduced into the subject's right naris. The olive was connected to a filter (Resp-Bac; Medicomp; Princeton, Minn.) and a respirator tube (AirLife; Allegiance Healthcare; McGaw Park, Ill.) with a gas-sampling connector and midstream sampling port (Respiratory Support Products; Irvine, Calif.) (figure 1). The subject closed his velum by holding his breath. Room air entered through the left nostril and was aspirated from the right nostril at a constant rate of 5 L/min. NO sampling was performed through a side port just distal to the tube.

On-line measurements. After 10 to 20 seconds of breath holding, a plateau of NO levels was reached. Extrapolation of the mean by software from this plateau was accepted as the on-line measurement value. For nasal NO measurements, 30 seconds of breath holding is accepted as sufficient to achieve a plateau at a flow between 3.2 and 5.2 L/min, (19) a fact that was confirmed in our study.

Off-line measurements. Four off-line samples were taken, beginning 5 to 10 minutes following the on-line measurement and after 20 seconds of breath holding. Samples were drawn through the side port and captured in four 50-mi syringes (adjustable-plunger-sealed gas/liquid syringes; Glenco; Houston). The syringes were sealed by closing a microvalve and twisting an adjustable plunger, and the samples were kept at room temperature.

The NO content in syringe #1 was measured immediately after it was drawn by connecting the syringe to the analyzer. The samples in syringes #2, #3, and #4 were analyzed 1, 2, and 4 hours later, respectively. (The 4-hour off-line measurements were made during only 18 of the 30 sessions). Upon the completion of the 30 sessions, a statistical analysis of the results was performed by correlation analysis and the Bland-Altman test. (22)

Results

All measurements were performed at a flow rate of 5 L/min. Results were reported as parts per billion (ppb) in accordance with the recommendation of the American Thoracic Society. (15) Correlations between on- and off-line measurements (as determined by linear regression analysis) and 95% confidence intervals were depicted in graph form; multiple correlation coefficient ([R.sup.2]) values ranged between 0.95 and 0.98 (figure 2). Bland-Altman plots depicted the difference to mean within 2 standard deviations (SDs) (figure 3). The differences were calculated by subtracting corresponding off-line measurements from on-line measurements. Agreement between datapoints was high, and only a few points were beyond the 2-SD threshold in any graph.

Discussion

The equipment currently used to measure NO in exhaled or nasal air is not portable, and transporting patients to an analyzer is not always feasible. In certain circumstances (e.g., when patients are hospitalized or when the distance to the laboratory makes it impractical to transfer the patient there), a more practical alternative is to store the air samples in suitable containers and carry them to the analyzer for delayed off-line measurement. Both Paredi et al (16) and Djupesland and Qian (17) have described methods of obtaining off-line measurements, but neither report identified a suitable and reliable container in which to collect and transport the samples. Paredi et al did suggest that there is no change in the NO concentration of samples kept in polyethylene reservoirs for up to 12 hours. To prevent the absorption of NO by water vapor and carbon dioxide ("quenching") over a 24-hour period, they placed silica gel into the reservoir.

The correlation between on-and off-line measurements in our study was high (95 to 98%). Therefore, we conclude that the storage media we used appear to have had a negligible effect on the chemical state of the NO-at least over a 4-hour period. The syringes used in our study were made of borosilicate glass, and the plungers were made of Teflon; neither of these materials reacts to most chemicals.

One possible problem with off-line measurement is diffusion of NO from or into the reservoir. The rate and direction of diffusion are related to the integrity of the reservoir's seal and the amount of ambient NO. If the concentration of ambient NO is less than the concentration of NO in the reservoir, diffusion from the reservoir to the ambient air can be expected. Conversely, if the concentration of ambient NO is greater, one would expect that the direction of diffusion would be toward the reservoir; however, such a diffusion is not possible in nasal samples because the NO in nasal air is always higher than the NO in ambient air. If the difference between the concentration of ambient NO and reservoir NO is great, the expected rate of diffusion will be high. The nasal air samples we kept in syringes for 4 hours did not undergo any dramatic change in NO concentration. Therefore, we conclude that the seals on our syringes were sufficiently tight to prevent leakage for at least 4 hours. A study to test the airti ghtness of these seals for longer durations is currently under way in our laboratory.

Finally, when performing off-line NO measurements in either a clinical or research setting, it is necessary to know the concentration of ambient NO in the environment in which the samples are taken. Therefore, the clinician or researcher must not neglect to sample the air in the surrounding environment in order to measure its ambient NO content.

Although this study was conducted with the assistance of only one subject, we tested our system with both low and high concentrations of nasal NO, and in every situation, the off-line measurement proved to be reliable. The results of our method of off-line measurement are reproducible and in good agreement with those seen with online measurement. We conclude that immediate and delayed off-line measurements of nasal NO are a practical complement to the on-line method.

[FIGURE 2 OMITTED]

Acknowledgments

The authors thank William Doyle, PhD, an otolaryngologist and director of the Rangos Research Center at the University of Pittsburgh, for his support of this research project. We also thank Kenwood Therapeutics of Fairfield, N.J., for providing the nasal olive tips.

References

(1.) Gustafsson LE, Leone AM, Persson MG, et al. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun 1991;181:852-7.

(2.) Lundberg JO. Airborne nitric oxide: Inflammatory marker and aerocrine messenger in man. Acta Physiol Scand Suppl 1996; 633:1-27.

(3.) Djupesland PG, Chatkin JM, Qian W, et al. Aerodynamic influences on nasal nitric oxide output measurements. Acta Otolaryngol 1999;119:479-85.

(4.) Arnal JF, Flores P, Rami J, et al. Nasal nitric oxide concentration in paranasal sinus inflammatory diseases. Eur Respir J 1999;13:307-12.

(5.) Sanders SP, Siekierski ES, Richards SM, et al. Rhinovirus infection induces expression of type 2 nitric oxide synthase in human respiratory epithelial cells in vitro and in vivo. J Allergy Clin Immunol 2001;107:235-43.

(6.) Jain B, Rubinstein I, Robbins RA, et al. Modulation of airway epithelial cell ciliary beat frequency by nitric oxide. Biochem Biophys Res Commun 1993;191:83-8.

(7.) Kim JW, Min YG, Rhee CS, et al. Regulation of mucociliary motility by nitric oxide and expression of nitric oxide synthase in the human sinus epithelial cells. Laryngoscope 2001;111:246-50.

(8.) Barnes PJ, Belvisi MG. Nitric oxide and lung disease. Thorax 1993;48:1034-43.

(9.) Lundberg JO, Settergren G, Gelinder S, et al. Inhalation of nasally derived nitric oxide modulates pulmonary function in humans. Acta Physiol Scand 1996;158:343-7.

(10.) Martin U, Bryden K, Devoy M, Howarth P. Increased levels of exhaled nitric oxide during nasal and oral breathing in subjects with seasonal rhinitis. J Allergy Clin Immunol 1996;97:768-72.

(11.) Lundberg JO, Nordvall SL, Weitzberg E, et al. Exhaled nitric oxide in paediatric asthma and cystic fibrosis. Arch Dis Child 1996;75:323-6.

(12.) Lundberg JO, Weitzberg E, Nordvall SL, et al. Primarily nasal origin of exhaled nitric oxide and absence in Kartagener's syndrome. Eur Respir J 1994;7:1501-4.

(13.) Baraldi E, Azzolin NM, Biban P, Zachello F. Effect of antibiotic therapy on nasal nitric oxide concentration in children with acute sinusitis. Am J Respir Crit Care Med 1997;155:1680-3.

(14.) Lindberg S, Cervin A, Runer T. Nitric oxide (NO) production in the upper airways is decreased in chronic sinusitis. Acta Otolaryngol 1997;117:l13-7.

(15.) American Thoracic Society. Recommendations for standardized procedures for the on-line and off-line measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and children--1999. Am J Respir Crit Care Med 1999;160:2104-17.

(16.) Paredi P, Loukides S, Ward S, et al. Exhalation flow and pressure-controlled reservoir collection of exhaled nitric oxide for remote and delayed analysis. Thorax 1998;53:775-9.

(17.) Djupesland PG, Qian A. A new method for off-line measurement of nasal and exhaled nitric oxide [abstract]. Allergy 2000;55(Suppl 63):42-3.

(18.) Silkoff PE, Chatkin J, Qian W, et al. Nasal nitric oxide: A comparison of measurement techniques. Am J Rhinol 1999;13:169-78.

(19.) Qian W, Djupesland PG, Chatkin JM, et al. Aspiration flow optimized for nasal nitric oxide measurement. Rhinology 1999;37:61-5.

(20.) Qian W, Chatkin JM, Djupesland PG, et al. Unilateral nasal nitric oxide measurement after nasal surgery. Ann Otol Rhinol Laryngol 2000;109:952-7.

(21.) Djupesland PG, Chatkin JM, Qian W, Haight JS. Nitric oxide in the nasal airway: A new dimension in otorhinolaryngology. Am J Otolaryngol 2001;22:19-32.

(22.) Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307-10.

From the Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh (Dr. Gungor), and the Department of Otolaryngology, Sisli Etfal Training and Research Hospital, Istanbul, Turkey (Dr. Vural).

Reprint requests: Anil Gungor, MD, Department of Pediatric Otolaryngology, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213-2583. Phone: [phone]; fax: [phone]; e-mail: [e-mail]