Nitric Oxide Breath Analysis: A Method
For Monitoring Inflammation In Asthma

Laurie Duckworth, RN; Niranjan Kissoon, M.D.; and Kevin Sullivan, M.D.
Laurie Duckworth, RN, BSN, CCRC, is the Clinical Research Coordinator, Nemours Children's Clinic. Niranjan Kissoon, M.D., FRCPC, FAAP, FCCM, is the Professor and Chief, Division of Pediatric Critical Care Medicine, University of Florida Health Science Center / Jacksonville. Kevin Sullivan, M.D. is a Pediatric Anesthesiologist and Intensivist, Nemours Children's Clinic.

Introduction

Asthma is a chronic inflammatory disorder of the airways resulting in airway hyperresponsiveness, reversible airway obstruction and symptoms such as wheezing, cough and shortness of breath. Asthma is the sixth ranking chronic condition in this country and the leading chronic illness in children. The disease is the number one cause of school absences. Annually 1.8 million patients visit our emergency rooms for asthma attacks. In 1998 asthma had an economic cost of 11.3 billion dollars to our nation.

When evaluating the asthmatic patient we currently attempt to monitor disease severity through clinical exam and pulmonary function test (PFT). Current tools to assess airway function include pulmonary function test (PFT) and peak flow meter measures, neither of which address airway inflammation. Procedures used for markers of airway inflammation include analysis of induced sputum, bronchoalveolar lavage, and bronchial biopsies. Obtaining adequate samples for sputum analysis can be difficult particularly in the pediatric patient. Bronchoalveolar lavage and bronchial biopsies are invasive, often impractical and are not without risks. Recently the NHLBI (National Heart Lung and Blood Institute) developed new guidelines for the management of asthma. One central theme in the new guidelines is to manage airway inflammation with early intervention. Recommendations include treatment with inhaled corticosteroids for patients who experience symptoms as little as two times per week.1 Unfortunately many times these guidelines are not followed by primary care physicians for a variety of reasons, one being the reluctance to use inhaled steroids for fear of growth suppression in the pediatric patient. In addition compliance with these types of medications is often unsuccessful. If however we had a tool that could measure inflammation, it is possible that medications could be titrated to achieve the desired clinical response.

There is mounting evidence to suggest that nitric oxide plays a key role in the physiological function of the airways and may serve as a suitable indicator of airway inflammation. The importance of this molecule is highly notable as the Nobel Prize was awarded this year to physicians and scientists who focused their research on nitric oxide.

Nitric Oxide -- Background

Endogenous nitric oxide is produced from the amino acid L- arginine by the enzyme NO synthase which has three isoforms.2 Two constitutive forms (cNOS) endothelial (eNOS) and neuronal (nNOS)) are small in quantity and serve basal metabolic functions. The third isoform, inducible (iNOS) is produced by inflammatory cytokines and endotoxin and plays a prominent role in asthma. All three isoforms are found in the human respiratory tract.3-4 We now know that nitric oxide serves a variety of pulmonary functions such as vascular and airway smooth muscle tone and is a mediator of the inflammatory response in the airway.5 Nitric oxide may also be necessary for ciliary action6 and is thought to aid in maintaining sterility in the lower respiratory tract due to its antimicrobial qualities against pathogens including viruses and mycobacteria.7

Exhaled Nitric Oxide

Gustaffson was the first to identify endogenous nitric oxide present in the exhaled air of animals and humans in 1991.8 Over the past several years numerous studies have been done describing increased exhaled nitric oxide in asthmatics as compared to healthy subjects.9-12 The production of iNOS is inhibited by glucocorticoids whereas cNOS is not.2,13

We also know that glucocorticoids inhibit induction of iNOS and reduce ENO levels in asthmatic patients.14 Therefore, measuring exhaled nitric oxide may be useful in assessing airway inflammation and monitoring the effectiveness and compliance of inhaled steroid medication in the asthmatic patient.

The exciting news is that measuring ENO is not difficult since breath sampling is noninvasive and practical. There are several analyzers now available commercially that have the capability to measure ENO. Most of the studies done to date measure nitric oxide using a chemiluminescence analyzer which detects the photochemical reaction between NO and the ozone in the analyzer. The beauty of this method is the ability to measure ENO directly in line to the analyzer or indirectly by obtaining an exhaled air sample in a balloon to be later analyzed at a more convenient time. Levels of NO in exhaled breath are reported in parts per billion (ppb) range. In our studies we use a Sievers 280 analyzer (Sievers Instruments, Boulder CO, USA) To date reported values for ENO have varied widely most likely due to significant differences in sampling technique. Major differences relate to exhalation flow rate and nasal contamination. The nasal sinuses excrete NO in very high concentrations of up to 23 parts per million (ppm).15

For the past year we have focused our research on determining the most effective method for analyzing and collecting ENO samples in the pediatric population. In our studies subjects inhale scrubbed room air quickly to total lung capacity. Nasal contamination is eliminated by having the subject exhale against a fixed resistance while maintaining a constant expiratory pressure that is displayed on a computer screen. Maintaining a fixed expiratory pressure ensures vellum closure preventing nasal NO contamination and provides a fixed controllable exhalation rate.16 The subject exhales until the ENO reaches a plateau, which usually takes approximately 3-5 seconds (Figure 1). Three samples are collected which have less than a 10% relative standard deviation. We have verified that ENO plateau levels increase as flow rate decreases.17-18 The easy nature of the collection has allowed us to obtain ENO samples with reproducibility in children as young as six years of age. This maneuver is as simple as if not easier to perform than routine pulmonary function testing.

duckwort.jpg (16139 bytes)
Figure 1.

Future Directions

How can monitoring exhaled nitric oxide enhance our ability to assess and monitor the asthmatic patient? Recent research has shown that exhaled nitric oxide may be a more sensitive marker for childhood asthmatic airway inflammation than serum levels of eosinophilic cationic protein or soluble interleukin-2 receptor.19

Commonly used tools to assess asthma include pulmonary function testing which are able to measure air flow but tell us little about the amount of underlying inflammation in the airway. With persuasive evidence to support findings that NO is increased in association with airway inflammation it seems reasonable to begin looking at ENO measurements for assessment of airway inflammation. The great advantage is that it is non-invasive and can be performed repeatedly in adult and pediatric patients regardless of airflow obstruction. Of concern is the expense of the chemiluminescence analyzer, although rapid advances in technology may decrease manufacturing cost in the near future. Barnes and Kharitonov have suggested that technological advances will make it possible to miniaturize these analyzers so they are portable and may be used at home in conjunction with peak flow meters.20 With these advances, measurement of ENO may soon make it to the bedside, the emergency room or the physicians office. Future research needs to look at the development of methods for ENO sampling in the infant and very young child. Defining the role of ENO in asthma takes on added significance when one considers that it may assist in determining the mechanism of leukotrienes in the inflammatory cascade. In addition, an appreciation of the role of nitric oxide may lead to new therapeutic agents for asthma for example, arginine analoges which may decrease nitric oxide production. Moreover, ENO may offer us a mechanism for close monitoring of steroid usage in order to prevent overdose and related growth concerns as well as monitor compliance.

Summary

Asthma is a major social and economic concern in the United States. Poorly controlled asthma causes hospitalization, urgent office visits, emergency room visits, missed days of work, limitations in activity which all contribute to the patients less than optimal quality of life. It is our responsibility as health care providers to find cost effective, acceptable, and practical ways to help our patients manage this disease that continues to increase in our community. With the NIH guidelines emphasis on early intervention and early anti-inflammatory treatment it is necessary to seek ways to quantify airway inflammation in the asthmatic patient. The ability to measure exhaled nitric oxide is promising as it may aid us in the diagnosis of asthma, adjustment of anti-inflammatory medications and monitoring of compliance with therapy.

REFERENCES

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  18. Kissoon N, Silkoff P, Murphy S Duckworth LD, Taylor C,. Effect of exhalation flow rates and inline versus gab sampling on pulmonary exhaled nitric oxide in children with asthma. 14th annual CNMC Symposium on ECMO & Advanced Therapies for Respiratory Failure. Keystone, Colorado, March 1-5, 1998 {Presentation}.
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November, 1999/ Jacksonville Medicine

 

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