232330 scient oms 03

232330 Scient Oms 03 05-04-21 08.00 Sida 3 Scientific Backgrounder, updated 2005
Produced by Aerocrine provider of NIOX® and NIOX MINO™
Exhaled Nitric Oxide
A Noninvasive Marker for Inflammation
232330 Scient Oms 03 05-04-26 12.41 Sida 4 Table of Contents
D. Detection of Steroid Unresponsiveness E. Prediction of Loss of Control of Asthma Emergency-Room Use C. Formation of Airway NO G. Safe Withdrawal of Inhaled Corticosteroids D. Relationship Between Airway Inflammation H. Dose Optimization and Asthma Symptoms Exhaled NO as an Early Marker of Asthma E. Relationship Between Exhaled NO and Atopy Exhaled NO in Epidemiology Measurement of Exhaled NO K. Air Pollution and NO Levels L. NO Levels and Occupational Health B. Reproducibility of Online NO Measurement Exhaled NO in COPD VII. Exhaled NO in Smokers D. Offline NO Measurement VIII. Exhaled NO in Other Diseases E. Normal Values for Exhaled NO A. Assessment of Chronic Cough F. Factors Influencng Levels of Exhaled NO B. Assessment of Cystic Fibrosis G. Exhaled NO in Asthma C. NO Levels and Transplantation H. Diurnal Variations Nasal NO Measurements Exhaled NO: Correlation with Known Inflammatory Markers A. Airway Hyper-Responsiveness B. Sputum Eosinophils D. Primary Ciliary Dyskinesia E. Cystic Fibrosis D. Neutrophilic Inflammation Flow-Independent Paramaters: Based on NO Diffusion Models F. Brochoalveolar A. NO Diffusion Models G. Pulmonary Function Exhaled NO in Asthma: Relationship to 2. Cystic Fibrosis A. NO and Corticosteroid Treatment 3. Allergic Alveolitis B. Combination Treatments C. NO and Anti-Leukotriene Treatment 5. Liver Cirrhosis D. NO and Other Anti-Inflammatory Treatment 6. Sjogren Syndrome E. Correlation with Disease Severity and Clinical Relevance of NO Measurements F. Quality of Life Clinical Use of Exhaled NO in Asthma XIII. Further Reading B. Response to Anti-Inflammatory Treatment A. Listed in Order of Appearance C. Monitoring Compliance with B. Listed in Alphabetical Order AERO003-Proof 04 7/4/05 5:37 pm Page 1 The Scientific Backgrounder discusses our current understanding of exhaled nitric oxide (NO) and its clinical utility, with emphasis on asthma.
This is the sixth edition of this publication, which now includes articles published up to the end of 2004 and some from early 2005. One of the most important recent developments is the finding that exhaled NO measurements are superior to the conventional clinical tests recommended by international guidelines in diagnosing asthma. In addition, further evidence is gathering to show that exhaled NO may be an early indicator of loss of asthma control or poor compliance, and that NO levels are powerful indicators of primary ciliary dyskinesia (PCD, nasal NO) and lung-transplant rejection (orally exhaled NO). This edition also includes, for the first time, sections on the relationship of NO with iNOS expression, air pollution, smoking and rhinovirus infection, and highlights the use of exhaled NO in Please note references that are new to this edition are highlighted in the text and the
NO is an important endogenous regulatory molecule that is widely distributed throughout the body. It acts as a messenger in many different biological processes, i.e. regulation of peripheral blood flow, platelet function, immune reactions and neurotransmission.1 NO elicits many of its physiological actions by activating cytosolic guanylate cyclase, which converts guanine triphosphate (GTP) to cyclic guanine monophosphate (cGMP). In 1998, Drs Robert F Furchgott, Louis J Ignarro and Ferid Murad were awarded the Nobel Prize for Medicine or Physiology for their basic discoveries in this field.
In biological tissues, NO is highly reactive, making direct determination of NO very difficult. Measurements have therefore often been indirect by determination of L-citrulline, which is produced when NO is formed from L-arginine, or by determination of nitrate and nitrite, which are the oxidized metabolites of NO. In the gas phase, NO is fairly stable at low concentrations and diffuses readily to nearby cells. Thus, when NO is formed in tissues and organs where it can diffuse into a lumen, it can be detected in gas samples collected from such organs.
Gustafsson et al. first reported in 1991 the detection of NO in exhaled air,2 and, soon after, Alving and coworkers found that NO in exhaled air was elevated in patients with asthma.3 There is now much evidence showing that measurement of the concentration of NO in exhaled air offers a useful non-invasive method of assessing inflammatory airway disease. AERO003-Proof 04 7/4/05 5:37 pm Page 2 Exhaled NO is not increased during bronchospasm unless there is coexisting inflammation. Therefore, exhaled NO may have a valuable role in differentiating between the inflammatory and bronchospastic components of clinical asthma, and may also be useful for guiding the therapeutic use of steroids and other anti-inflammatory agents.4 This document concludes that in asthma: • the concentration of exhaled NO correlates well with inflammatory markers in bronchial biopsies and bronchial lavage (i.e. eosinophilic concentration) • corticosteroid therapy reduces exhaled NO in a dose-dependent manner • exhaled NO levels correlate with markers of disease control • exhaled NO is a sensitive measure of airway inflammation that reacts rapidly in response to treatment or exacerbation of disease • clinical applications of exhaled NO measurement include monitoring compliance and response to treatment, disease activity, and the prediction of acute exacerbations • NO measurements are superior to conventional clinical tests as recommended by international guidelines in the diagnosis of asthma • simple and sensitive methods have been developed to determine exhaled NO • measurement of exhaled NO is non-invasive, easy, and convenient for patients • normal values at 50 mL/s are being established.
Formation of Airway NO
The synthesis of NO is mediated by NO synthases (NOS), which exist in constitutive (cNOS) and inducible (iNOS) isoforms. Constitutively expressed NOS are present particularly in endothelial cells (eNOS) and neural tissue (nNOS). All three forms of NOS, i.e. eNOS, nNOS and iNOS, have been shown to be present in the airways.5 However, only the expression of iNOS correlates with levels of exhaled NO.6 Expression of iNOS is seen particularly in airway epithelial cells and the expression is markedly increased in asthma.7,8 Nitric oxide formed by cNOS may lead to cGMP-dependent relaxation of airway smooth muscle, whereas high quantities of NO released by iNOS may be associated with pro-inflammatory effects.9,10 In addition, endothelial NOS has been localized to the bronchial and pulmonary circulation and a known DNA sequence variant in eNOS is associated with decreased levels of exhaled NO.11,12 Guo and colleagues demonstrated that patients with asthma exhibit increased expression of iNOS mRNA in airway epithelium compared with healthy controls, but iNOS mRNA was not detected in alveolar macrophages.13 Moreover, those patients who were receiving corticosteroids had decreased expression of iNOS protein and mRNA compared with those not receiving this treatment. iNOS expression can be induced by a variety of stimuli, in particular inflammatory cytokines, but the exact mechanism is not understood even though it has been suggested that AERO003-Proof 04 7/4/05 5:37 pm Page 3 the transcription factor STAT-1 is involved (Figure 1).13 Another important transcription factor, NF-kappa B, is upregulated in allergic inflammation and downregulated by corticosteroids.14 This transcription factor is essential for expression of, for example, eotaxin and granulocyte- macrophage colony-stimulating factor (GM-CSF). These proteins are also expressed in bronchial epithelial cells in asthma. It seems that STAT-1 is upregulated simultaneously with, or possibly as a consequence of, the upregulation of NF-kappa B in the asthmatic human airway epithelium. Some studies indicate that iNOS also plays an important role in pulmonary inflammation.
For example, Cuzzocrea and co-workers have shown that iNOS-knockout mice have a less severe pulmonary inflammatory response to carrageenan than wild-type mice.15 Oral treatment with a selective iNOS inhibitor has been shown to reduces exhaled NO by 80–90% in both patients with asthma and healthy controls.16 Basal iNOS expression Induced iNOS expression Th2-driven (allergic) inflammation Figure 1. Possible mechanism of how airway lung inflammation leads to increasedlevels of NO in exhaled air. Courtesy of Prof. Kjell Alving Relationship Between Airway Inflammation and Asthma
Symptoms
Asthma is a chronic inflammatory disorder of airways. But how are NO and inflammation related to the main symptoms of asthma? The current concept of asthma pathogenesis involves a chronic inflammatory process, which causes the development of airflow limitation and increased responsiveness to allergens. The airway inflammation is characterized by an increased number of activated eosinophils, mast cells and T-lymphocytes in the airway mucosa and lumen. The actions of these cells result in epithelial damage, swelling, mucus secretion and airway smooth muscle contraction. Thus, inflammatory cells and mediators are the cause of asthma symptoms. For AERO003-Proof 04 7/4/05 5:37 pm Page 4 example, Amin and co-workers showed that the concentration of eosinophils correlated inversely with epithelial integrity of the airways in a group of individuals with atopic asthma, indicating that inflammation plays an important role in airway remodelling.17 It has been shown that patients with asthma experience an early and late inflammatory response when challenged with allergens.18 The early response is transient and is characterized by an increase in neutrophils. In contrast, the late response is sustained and is associated with an increase in eosinophils and T-cells. It is generally considered that it is this late response that is associated with an increase in NO levels. However, one study concluded that neutrophils also contribute to NO production in asthma.19 Furthermore, exhaled NO levels were found to be similar at baseline in patients who exhibited an isolated early response after challenge and those who had both an early and late response.20 In this study, allergen challenge resulted in an increase in NO levels only in the group who had both an early and late response, although the increase was not significant.
NO has a wide-ranging role in mammalian physiology as a messenger molecule. It is a potent smooth muscle relaxant, a neurotransmitter in the central and peripheral nervous system, and is involved in hormone release. It is thought that NO may be important in regulating blood flow in the lungs and airways and, thus, dysregulation of NO production may have a role in the pathogenesis of asthma.21 Sputum nitrite/nitrate (µM) Levels of NO derivatives in induced sputum have been shown to correlate with Figure 2. The concentration of nitrites and nitrates in the ratio of airway wall thickness to lumen sputum correlates with the ratio of airway wall thickness(T ) to lumen diameter (DL) in asthmatic patients ( r = 0.9; diameter in adults with asthma, suggesting a role for NO in airway remodelling Using sensitized rats, Chiba et al. showed that antigen-induced airway hyper- responsiveness is associated with increased generation of NO, probably as a result of iNOS activity in epithelial and infiltrating inflammatory cells.23 At least two studies have investigated the link between exercise-induced bronchoconstriction and NO in patients with asthma.24,25 In one of these studies, patients who experienced exercise-induced bronchoconstriction had higher concentrations of NO derivatives in their sputum than other asthma patients.24 Moreover, excess NO production appeared to contribute to the prolonged airway narrowing stimulated by exercise in susceptible patients. These findings are supported AERO003-Proof 04 7/4/05 5:37 pm Page 5 in the other study, in which inhibition of NO synthesis reduced airway constriction following challenge.25 The authors conclude that NO plays an important role in the pathogenesis of In summary, the symptoms of asthma are now known to result from the actions of eosinophils and other inflammatory cells. Increased NO levels are associated with eosinophilic activity, but the exact role of NO in the development of asthma symptoms remains unknown. Relationship Between Exhaled NO and Atopy
When assessing the relationship between NO and asthma, one must consider the role of atopy. It is known that atopy is a genetically determined condition and is a frequent characteristic of asthma. However, atopic individuals do not necessarily develop asthma, and asthma patients without atopy are not rare.
Several studies have shown that patients with atopic asthma have higher levels of exhaled NO than other patients with asthma.26–32 Indeed, some authors report no difference in exhaled NO levels between non-atopic asthma patients and healthy controls.26,29 Moreover, there is evidence that atopic individuals without asthma have abnormally high NO levels. For example, Horváth and Barnes showed that healthy, non-smoking individuals who were atopic had significantly higher exhaled NO levels than those who were non-atopic.33 Other studies have shown a correlation between exhaled NO levels and skin-prick test reactivity to house dust mite.34,35 A study of 450 children by van Amsterdam and colleagues showed that sensitization to indoor allergens was associated with higher levels of exhaled NO (1.5X) compared to non-sensitized children (P < 0.05; Figure 3).35 Leuppi et al. have shown that sensitization to house dust mites was associated with raised exhaled NO levels in the winter season. The NO values correlated significantly with airway hyper- responsiveness to histamine, independently of whether the children had symptoms or not.36 This was also shown by Langley et al. who demonstrated higher exhaled NO levels in patients who had both been sensitized and exposed to high levels of indoor sensitizing allergen correlating with a more severe form of the disease.37 More recently, this relationship between exhaled NO and airway responsiveness has been shown to be Figure 3. Sensitization to indoor allergens is associatedwith higher levels of exhaled NO compared to non- evident only in atopic children.31,38 It was sensitized children35 AERO003-Proof 04 7/4/05 5:37 pm Page 6 shown by van Amsterdam et al. that inhalation of grass pollen increased the level of exhaled NO in children sensitized to grass pollen.30 In a study involving patients with persistent rhinitis, sensitization to pollen was associated with seasonal variation in exhaled NO levels.39 A strong correlation between exhaled NO and pollen counts in the 2 weeks before NO measurement has been shown in patients with seasonal allergic asthma (Table 1).40 The results suggest that NO levels are significantly increased 8–14 days after pollen exposure.
Such evidence has led some experts to conclude that increased NO levels are a feature of atopy rather than asthma. Raised exhaled NO levels appear to be associated with an underlying mechanism linking atopy and airway responsiveness but not necessarily The evidence linking atopy and increased NO levels is, however, controversial. Gratziou et al. reported no difference in exhaled NO levels between atopic and non-atopic healthy Table 1. Correlation between exhaled NO levels (measured at 250mL/s) and mean pollen
count over different time periods before NO measurement40
Time period relative to day of NO measurement
Correlation between NO and pollen count
Regression coefficient (95% CI) 0.0089 (0.004–0.013) Regression coefficient (95% CI) 0.0129 (0.0086–0.0173) Regression coefficient (95% CI) 0.0132 (0.0090–0.0174) Regression coefficient (95% CI) 0.0120 (0.0077–0.0163) Regression coefficient (95% CI) 0.0146 (0.0096–0.0196) Regression coefficient (95% CI) 0.0112 (0.0061–0.0163) Regression coefficient (95% CI) 0.0144 (0.0103–0.0186) Regression coefficient (95% CI) 0.0131 (0.0088–0.0173) Day –8 to –14 Regression coefficient (95% CI) 0.0081 (0.0041–0.0121) AERO003-Proof 04 7/4/05 5:37 pm Page 7 individuals.27 Similar results were described by Berlyne and co-workers.42 Important insights were published by Olin and co-workers, who showed that only atopic patients who had recently been exposed to the relevant allergen had elevated levels of exhaled NO. Atopic patients who had not been exposed to a relevant allergen or who had never experienced symptoms of asthma or rhinitis showed normal eNO.43,44 Gratziou et al. reported that allergic rhinitis is associated with increased NO.27 However, Lopuhaa and colleagues showed that although baseline exhaled NO is significantly lower in non-asthmatic rhinitis compared with asthma (P < 0.006), the difference in exhaled NO at baseline is abolished after allergen exposure, due to a significantly greater increase in exhaled NO in non-asthmatic rhinitis.45 These findings underline the similarities in bronchial changes in allergic patients with and without asthma.
In considering this issue, one must remember that exhaled NO is a marker for inflammation. Thus, it is likely that increased exhaled NO levels in an atopic individual with rhinitis indicate general airway inflammation and possibly an increased risk of developing Airway inflammation in asthma may represent a favourable environment for respiratory viral infections, augmenting virus-induced exacerbations in asthma. de Kluijver et al. showed that exhaled NO increased significantly during allergen exposure (P < 0.001), whereas it did not change significantly after RV16 infection (P = 0.8) or successive allergen exposure and RV16 infection (P = 0.9; Figure 4).46 Results show that repeated low-dose allergen exposure and RV16 infection induce distinct inflammatory profiles within the airways in asthma without apparent interaction between these two environmental triggers. This suggests that Allergen + placeboPlacebo + RV16 Allergen + RV16 mean (± SE) (ppb) Allergen vs. placebo P ≤ 0.001RV16 vs. placebo Figure 4. Exhaled NO increased significantly (P < 0.001) during allergen exposurecompared with placebo, but did not change significantly after RV16 infection or successiveallergen exposure and RV16 infection46 AERO003-Proof 04 7/4/05 5:37 pm Page 8 priming airways with repeated low-dose allergen does not result in an aggravated response with regard to airways obstruction and airway inflammation after RV16 infection in patients with mild asthma.
These studies indicate that exhaled NO is a marker of increased inflammation activity triggered by allergen exposure. However, further investigations are still required to determine fully the relationship between NO, atopy and the development of asthma.
Measurement of Exhaled NO
The most widely used method for measurement of exhaled NO is chemiluminescence after reaction with ozone, which allows measurements down to approximately 1 part per billion (ppb).47 Recently, however, a new sensor technology has been applied to allow the measurement of exhaled NO using a hand- held device suitable for routine clinical [NO] in orally-exhaled air (ppb) practice.48 As NO is continuously formed in the airways, the concentration of NO 100 150 200 250 300 350 400 will vary with the flow of exhaled air Exhalation flow rate (mL/s) (Figure 5), a fact that has been well documented by Silkoff et al.49 Kissoon and Figure 5. Relationship between exhalation flow rate andNO concentration in orally exhaled air from a healthy colleagues have measured exhaled NO levels at a series of low flow rates (4, 5, 7, 10, 15, 23, 31 and 46 mL/s) and documented changes in the concentration (101.3, 87.7, 81.1, 62.1, 74.2, 62.3, 46.4, and 36.9 ppb, respectively).50 It is therefore important to register the flow rate if NO is expressed as a concentration. The flow rate recommended in 1997 by a task force of the European Respiratory Society (ERS) was quite high (10–15 L/min or 167–250 mL/s).51 Most authors have used about 100 mL/s. Since the publication of the ATS guidelines in December 1999, most authors have been using the recommended 50 mL/s.52 Higher or lower flow rates may be used in certain situations, but the flow rate should always be recorded. Exhaled NO is usually determined during single-breath exhalations. The recommended technique for adult patients involves inspiration of NO-free air via a mouthpiece to total lung AERO003-Proof 04 7/4/05 5:37 pm Page 9 capacity, followed immediately by full exhalation at an even rate through the mouthpiece into the apparatus.52 During exhalation, an excess pressure is created in the oral cavity, which ensures closure of the velum and prevents contamination of the sample with nasal air. This is important as nasal air contains high concentrations of NO, probably derived from paranasal sinuses Sinus sampling(1000–30000 ppb) Nasal breathing (15–40 ppb) Oral breathing (5–15 ppb) Tracheal breathing (< 3 ppb) Figure 6. Schematic drawing showing NO levels measured by chemiluminescence at different levels of the respiratorytract. The subjects were breathing normal tidal volumes through the nose, mouth or through a permanenttracheostomy. Sinus air was aspirated through a catheter placed in the maxillary sinus of healthy subjects. Courtesy ofJon Lundberg, MD Contamination from the oral cavity has been identified as another potential source of error in the determination of exhaled NO; it may be avoided by special mouthwash procedures.55 NO levels in ambient air may be high and have been shown to influence the measurement of exhaled NO,56 although some investigators have not confirmed this.57 It is therefore recommended that NO-free air (< 5 ppb) is inhaled, which can be achieved, for example, by absorption of NO in a scrubber. In the short term, spirometry, hyper-responsiveness test and sputum induction appear to reduce exhaled NO levels. Therefore, NO should be measured prior to these tests.58–62 Reproducibility of Online NO Measurement
A pre-requisite for the routine clinical use of exhaled NO in asthma management is that the method shows good reproducibility. Kharitonov and colleagues have shown that highly reproducible measurements can be obtained using the NIOX® NO monitoring system.63 The study demonstrated that the mean standard deviation in NO measurements was 2.1 ppb, suggesting that a change in exhaled NO of 4 ppb is likely to result from a change in the status AERO003-Proof 04 7/4/05 5:37 pm Page 10 of the inflammation (Figure 7). In another study, the mean standard deviation of NIOX® was shown to be < 2.0 ppb.64 In this study, data from NIOX® were compared with data from NIOX MINO™ – the new hand-held exhaled NO analyser. An excellent correlation (r = 0.98; P < 0.001) was seen between the results from the two devices, with the mean standard deviation Distance from mean in session (ppb) –6 in NO measurements with NIOX MINO™ being < 2.5 ppb.
Mean exhaled NO in session (ppb) Figure 7. Bland-Altman analysis for the repeatability of It should be noted that intrapatient and fractional exhaled NO values (n = 637 measurements).63Measured with NIOX® interpatient variability can affect levels of exhaled NO.65,66 Exhaled NO levels may also vary over time in patients with stable asthma.67 Although the single-breath method is the most reliable way of measuring exhaled NO, the breathing manoeuvre required is too complicated to perform for very young children. Many groups have tried to overcome difficulties by devising methods for measuring exhaled NO from tidal breathing, which requires only passive cooperation. Visser and colleagues developed an offline method of measuring NO in mixed exhaled gas collected during 5 minutes of tidal breathing in children 4–14 years of age.68 This method required children to breathe quietly through a mouthpiece, whilst wearing a noseclip.69 NO- free air was inhaled from a Douglas bag and exhaled gas was collected for 5 P < 0.05 P < 0.01 minutes through a non-rebreathing valve into a separate Douglas bag. Using this method, the group showed that exhaled P < 0.001 NO levels are significantly elevated in asthmatic children compared to non- asthmatic children (Figure 8), in agreement with other studies.70–72 Although other groups have used tidal breathing methods,26,73–75 this study was the first to 0 Non-asthmatic Asthma collect mixed exhaled air, thus obtaining a mean NO concentration of several breaths and eliminating potential influences of Figure 8. Exhaled NO levels are higher in asthmaticchildren compared with non-asthmatic controls and flow changes and breath.76 asthmatics on inhaled corticosteroid therapy68 AERO003-Proof 04 7/4/05 5:37 pm Page 11 Tidal measurements have been used successfully to differentiate steroid-naïve young children with intermittent asthma from healthy children, from non-asthmatic children with chronic cough, and from asthmatic children treated with inhaled steroids.77 However, recent evidence suggests that offline tidal measurements cannot distinguish patients with wheeze and healthy controls.78 Furthermore, many factors appear to affect these measurements.79 An online method of measuring NO during controlled tidal breathing has been described.80 Resistance at the exhalation valve was continuously adjusted by the operator with the aim of controlling the exhalation flow. Online tidal measurements have been used successfully to show that exhaled NO levels fall significantly after corticosteroid treatment.81 However, although promising, wider use of online methods would require dedicated software to adjust for the lag time (staggering of flow and NO signal) and the individual rinse volume. In addition, any contamination from nasal air would not be apparent as the NO profiles are not monitored. Reports suggest that online tidal NO measurements are not able to discriminate effectively between asthmatic and healthy children.82,83 Offline NO Measurement
The online, single-breath measurements described earlier are limited to institutions because of the size of the equipment required. Offline methods to collect exhaled air to send to a clinic or laboratory for determination of NO are under investigation.83,84 Offline collection of samples for NO estimation offers the potential for samples to be collected at sites remote from the analyser, but it also carries potential disadvantages. These include contamination with gas not derived from the lower airway, sample deterioration during storage and transportation, and the inability to offer immediate feedback and assessment of technique.52 The ATS recommendations addressed offline measurement of NO.52 Critical considerations for offline analysis include the use of NO-free air for inspiration and the recording of the expiratory flow rate. Kissoon et al. developed a method whereby online sampling and offline sampling could be done simultaneously at various flow rates, in order to compare NO measurements using the two techniques.50 They found a strong correlation between offline and online measurements of NO with flow rates of 46, 31, 23, and 15 mL/s (P < 0.001). Similarly, Jöbsis and colleagues showed a good correlation between offline and online techniques at a flow rate of 50 mL/s, provided that the first 220 mL of the offline exhalation was excluded.85 Discarding the first 220 mL of the exhalation reduced contamination from ‘dead space' air.
Other publications have suggested that the use of a prebag to collect exhaled air will initially reduce contamination from ambient air.86 It has been shown that the use of a 1 L prebag may prevent such contamination. However, the use of an NO scrubber is preferred with regard to the reduction of ambient air contamination. AERO003-Proof 04 7/4/05 5:37 pm Page 12 Table 2. Normal values for exhaled single breath NO measured at a flow rate of 50 mL/s
Exhaled NO (ppb)
Jöbsis et al., 200185 • Online method • Offline method Kharitonov et al., 200363 • Measurements in morning and afternoon Kissoon et al., 200050 • Flow rate of 46 mL/s Pedroletti et al., 200087 • Flow rate of 48 mL/s Pijnenburg et al., 200288 • Offline method 14 female children Scollo et al., 200089 Malmberg et al.,200390 Joaville et al.,200391 • Flow rate 100 mL/s Buchvald et al., 200492 Chatkin et al., 199993 • Flow rate of 45 mL/s Foresi et al., 200094 • Flow rate of 47 mL/s Kharitonov et al., 200363 • Measurements on 5 consecutive days. On the last day,measurements were madeat 4 different time points Malerba et al., 200195 Olin et al., 200096 Olin et al., 200144 • NO measured outside – without rhinitis Non-atopic– without rhinitis Papi et al., 200097 • Flow rate 67–83 mL/s Sandrini et al., 200398 • Flow rate 46 mL/s ElHalawani et al., 200399 Tornberg et al., 2003100 • All female patients Normal Values for Exhaled NO
Using the chemiluminescence technique, several laboratories have reported normal values for exhaled NO. The values reported initially were quite high and variable in some cases, which may reflect a lack of standardization at that time, particularly relating to the exhalation flow rate. Data obtained according to the ATS guidelines have now appeared at major conferences and in the literature. These values show much greater consistency (Table 2). The studies show that healthy individuals usually have exhaled NO values of between 10 and 20 ppb (children slightly lower, 5–15 ppb), if measured according to ATS guidelines. Analysis of the variation seen in such studies, suggests that 97% of healthy individuals have NO levels of less than 35 ppb (< 25 ppb in children).
AERO003-Proof 04 7/4/05 5:37 pm Page 13 Factors Influencing Levels of Exhaled NO
Airway inflammation and asthma appear to be the most important causes of increased levels of exhaled NO. Treatment with inhaled corticosteroids reduces the amount of exhaled NO (see Section IV Exhaled NO in Asthma: Relationship to Anti-Inflammatory Treatment). Other factors that have been demonstrated to decrease or increase the levels of exhaled ↓ = decrease in level, ↑ = increase in level • Hypertension101,102 • Pulmonary hypertension103,104 • Ciliary dyskinesia105–109 ↓ (particularly nasal NO) • Airway viral infection112,113 • Allergic rhinitis44,46,91 • Alveolitis114,115 • Lung transplant rejection (bronchiolitis obliterans syndrome [BOS])116–119 • COPD97,120–122 • Pulmonary sarcoidosis123,124 • Chronic bronchitis120 • Chronic cough77,125–127 • Systemic sclerosis103,128 • Idiopathic pneumonia syndrome131 • Cystic fibrosis132,133–13 ↓, but ↑ during respiratory exacerbation • Premenstrual asthma136 • Nasal polyposis138 • Chronic lung disease139,140 ↑ in infants, but ↓ in school children • Sickle cell anaemia141–143 • Passive smoking148,149,150 • Spirometric manoeuvres62 • Sputum induction58,152 • Caffeine153,154 ↓, no change in patients with asthma • Nitrate-rich diet155 • Alcohol consumption156,157 small ↓ in healthy individuals, possibly larger ↓ in patients with asthma • Cardiac pulmonary bypass158 • Exercise99,159–162 *Only a selection of articles are included that typify results.
AERO003-Proof 04 7/4/05 5:37 pm Page 14 The finding that NO levels are reduced immediately after bronchoconstriction caused by histamine or methacholine challenge shows that hyper-responsiveness tests should be performed after NO measurements.59,151 Exhaled NO levels appear to be influenced by the size of the airways, and, therefore, some differences between males and females, and between children and adults, have been reported.163,164 Another interesting finding is that NO levels increase in the period after a nitrate-rich meal is eaten.155 Although further studies are needed to confirm this finding, physicians may consider recommending avoidance of high-nitrate foods before measurement of NO levels.
Exhaled NO in Asthma
Alving et al. were the first investigators to report increased levels of exhaled NO in asthma.3 Eight atopic patients with documented allergy towards at least rat allergen, and with mild symptoms of asthma and rhinitis, had two- to three-fold increased NO levels compared with 12 healthy, non-smoking control individuals. These findings were soon confirmed by Kharitonov et al.165 They reported much higher levels of exhaled NO, but the ratio between normal subjects (n = 67) and asthmatic patients (n = 52) was very similar, with 3.5-fold higher concentrations in the asthmatic patients. A relationship between an increase in exhaled NO and allergen exposure was also documented by Kharitonov et al.166 As determination of exhaled NO is easy and non-invasive, the method appears to be particularly attractive for paediatric use, and several authors have studied NO levels in children with asthma.26,56,72 Piacentini et al. demonstrated an increase in exhaled NO following allergen exposure in asthmatic children; the increase was effectively prevented by treatment with inhaled corticosteroids.167 Following the publications by Alving et al.3 and Kharitonov et al.165,166 there have been many reports confirming the increased levels of NO in exhaled air from patients with asthma.
Table 3 lists some examples. Most of these studies have found NO to be elevated by two- to four-fold compared with matched controls. Considering the data presented in Table 3 and other studies that have used flow rates of 50 mL/s,44,168 asthma patients tend to have exhaled NO values between 25 and 80 ppb if measured according to ATS guidelines; higher values may occur in some patients, particularly those with exacerbations.
AERO003-Proof 04 7/4/05 5:37 pm Page 15 Table 3. Exhaled NO in asthma.
Flow rate
Exhaled NO (ppb)
Kharitonov et al., 200363 24.9 ± 22.3 (children) Olin et al., 200096 Kharitonov et al., 200363 61.7 ± 48.4 (adults) Scollo et al., 200089 76.2 ± 26.2 (children) Malmberg et al., 200390 22.1 ± 3.4 (children) Sacco et al., 200332 Silvestri et al., 2003169 Smith et al., 2004170 52.0 ± 34.0 (both) Diurnal Variations
Diurnal variation of exhaled NO in patients with asthma appears to be small, even in patients with nocturnal asthma.171 Georges et al. found that exhaled NO was slightly but significantly lower during the night than during the day in patients with nocturnal asthma.172 The mean value of 77.2 ± 8.2 ppb fell to 68.4 ± 8.7 ppb at 22:00 h and to 66.0 ± 8.5 ppb at 04:00 h.
Patients with non-nocturnal asthma did not show any significant diurnal variation. In another study, Mattes et al. demonstrated a cosine-like circadian rhythm with lowest levels at 19:00 h and highest at 07:00 h, when exhaled NO was measured every third hour.173 In another study involving patients with nocturnal asthma, iNOS levels in bronchial biopsies were shown to increase during the day, but not during the night.174 More recently, Kharitonov and coworkers examined exhaled NO levels in healthy and asthmatic children and adults, which showed no evidence of diurnal variation.63 AERO003-Proof 04 7/4/05 5:37 pm Page 16 Exhaled NO: Correlation with Known
It is likely that changes in the levels of different inflammatory markers after corticosteroid treatment reflect the influence of steroids on different mediators of the inflammatory response.175 Exhaled NO reflects airway inflammation, supported by studies correlating NO levels with conventional markers of airway inflammation.30,169,176,177 The correlations reported are often only moderate or weak, but this is to be expected as the different markers reflect different aspects of inflammation. More importantly, recent evidence has shown that exhaled NO levels correlate with the results from examinations of bronchial biopsies and bronchoalveolar lavage (BAL) – the ‘gold standard' assessment of airway inflammation.178–180 Compared with procedures such as BAL and airway biopsy, measurement of NO is non- invasive, safe, and causes no inconvenience to the patient. Airway hyper-responsiveness is measured as the concentration of inhaled bronchoconstrictor agent required to produce a 20% drop in forced expiratory volume in 1 second (FEV [PC ]).
Conflicting evidence regarding the correlation between NO levels and hyper-responsiveness has been reported. However, a good correlation between exhaled NO and bronchial hyper- reactivity has been shown by many investigators. Al-Ali and Howarth demonstrated a relationship between exhaled NO levels and the FEV (PC ) dose of inhaled histamine (compared with post-saline FEV ) in 26 non-smoking, atopic asthmatic patients with a mean age Rank of exhaled NO of 27 years (Figure 9).181 Salome et al. also reported a significant correlation between exhaled NO and hyper-responsiveness to Figure 9. Relationship between exhaled NO levels and histamine in young adults.182 PC histamine daily ( r = –0.51, P = 0.008)181 Similar relationships between exhaled NO and airway hyper-responsiveness have been demonstrated by other groups125,183–187 who used methacholine rather than histamine as the bronchoconstrictor. In addition, a correlation has been reported between NO and hyper- responsiveness to saline.38,188 Lúdvíksdóttir et al. measured exhaled NO in a group of atopic and non-atopic asthmatic patients, and healthy controls.28 Exhaled NO was elevated only in atopic patients, and in this group, it correlated well with the airway hyper-responsiveness to methacholine. In non- atopic patients and controls, there was no relationship between airway hyper-responsiveness AERO003-Proof 04 7/4/05 5:37 pm Page 17 Many studies show no correlation between exhaled NO levels and airway hyper- responsiveness. In a study of children with mild intermittent asthma, Silvestri et al. were unable to find a correlation between exhaled NO and hyper-responsiveness.189 Olin and co- workers also reported no correlation with methacholine hyper-responsiveness in pulp mill workers exposed to ozone.190 In addition, adults with mild asthma have been reported to show no correlation between NO and hyper-responsiveness.175 A study by van den Toorn and colleagues failed to show a significant correlation between hyper-responsiveness to methacholine and exhaled NO in patients with ongoing asthma and those in clinical remission.191 There was, however, a significant relationship between exhaled NO and hyper-responsiveness when adenosine-5'-monophosphate (AMP) was used.
It has been shown by de Meer et al. that hyper-responsiveness to AMP tends to correlate with serum eosinophilia, whereas responsiveness to methacholine correlates with FEV , indicating that the former is a better marker of inflammation.192 Several studies support the theory that the bronchial response to AMP is more closely associated with airway inflammation than the response to direct bronchoconstrictors such as histamine or methacholine.3,193 A recent study in asthmatic disease patients in whom disease was already stabilized and well controlled by use of inhaled corticosteroids, showed that both bronchoconstriction in response to AMP and increased exhaled NO levels were significant predictors for failure in inhaled corticosteroid dose reduction.194 Therefore, determination of AMP responsiveness and exhaled NO levels may be useful in identifying which patients might deteriorate when their dose of inhaled corticosteroid is reduced.
Grönke et al. found that there was a significant correlation between NO levels and hyper- responsiveness to methacholine in patients who have had asthma for 16 or fewer years, but that this correlation was not present in patients who have had asthma for longer duration.195 Importantly both Grönke and de Meer's groups showed a significant correlation between NO levels and sputum eosinophils, suggesting that methacholine hyper-responsiveness in patients with a long duration of asthma does not reflect active eosinophilic ElHalawani and colleagues examined whether exhaled NO levels before or after exercise could be used as a surrogate marker of exertional bronchoconstriction in a population referred specifically for the evaluation of exercise-induced broncho- Figure 10. Scatterplot of exhaled NO and exercise-inducedbronchoconstriction (EIB). Sensitivity = 1.0; specificity = constriction.99 They showed that no patient 0.31; positive predictive value = 0.19; negative predictivevalue = 1.0. No one with a baseline exhaled NO < 12 ppb with very low pre-exercise exhaled NO demonstrated EIB99 AERO003-Proof 04 7/4/05 5:37 pm Page 18 levels (< 12 ppb) demonstrated bronchial hyper-responsiveness to exercise. Hence exhaled NO measurements may obviate the need for bronchoprovocation testing in patients who complain of exertional dyspnoea (Figure 10).
Eosinophilic inflammation is a hallmark of bronchial asthma.196 Two studies by Jatakanon et al.184,197 have demonstrated a relationship between exhaled NO and the fraction of eosinophils in induced sputum. The first study measured sputum eosinophils and exhaled NO in a group of stable asthmatics maintained on β -agonists alone, and found a significant correlation between the two parameters.184 The second study examined a group of patients in whom mild exacerbations of asthma were induced by reducing the dose of inhaled maintenance steroids.197 In those patients who developed an exacerbation of asthma, increases in both sputum eosinophils and exhaled NO were significantly correlated with deterioration in airway function. In addition, changes from baseline in exhaled NO levels and the concentration of sputum eosinophils were greater in patients after they had experienced an exacerbation compared Figure 11. Effect of exacerbation of asthma on changes in with the changes seen in those who did not markers of airway inflammation.197 Peak expiratory flow experience an exacerbation (Figure 11).
= PEF; change in peak-flow variability = PFvar There was also a significant correlation between the change in exhaled NO and the change in FEV and the amount of rescue bronchodilator required.
Other studies have drawn similar conclusions. Piacentini et al.198 found that exhaled NO correlated with sputum eosinophils, particularly in steroid-naïve patients, and Mattes et al.
reported a correlation between markers of eosinophilic airway inflammation in children with A small study of steroid-naïve non-atopic asthmatic patients showed that levels of sputum eosinophils were positively correlated with dose of beclomethasone dipropionate (BDP) inhaled, indicating that the monitoring of eosinophilic airway inflammation may also be useful in the assessment of the effects of inhaled steroids in patients without history of AERO003-Proof 04 7/4/05 5:37 pm Page 19 Measurement of sputum eosinophilia requires induction of sputum and is an inconvenient and perhaps hazardous procedure for both adults and children. It may be more convenient to measure the r = 0.63, P < 0.01 eosinophil count in peripheral blood, which correlates well with the degree of Eosinophils (n) allergic sensitization in children.201 A statistically significant correlation between both exhaled NO and total number of blood eosinophils and percentage of blood eosinophils in children with atopic asthma r = 0.63, P < 0.01 has been demonstrated by Silvestri et al.
Figure 12. Correlation between NO levels in orally In a study of children with exhaled air and blood eosinophilia in asthmatic patientstreated with inhaled β -agonists on an as-necessary basis.
exacerbations of asthma, Lanz et al. found (a) Number of eosinophils; (b) percentage of eosinophils202 that exhaled NO was a more sensitive marker of disease activity than either serum eosinophilic cationic protein (ECP) or soluble interleukin-2 receptor (sIL2R).203 Furthermore, repeated measurements after treatment suggested that exhaled NO was a more useful indicator of response to corticosteroid therapy than either serum ECP or sIL2R. Crater et al. found a highly significant correlation between exhaled NO and peripheral blood eosinophilia in adult patients with acute and stable asthma.204 A combination of exhaled NO > 10 ppb and eosinophilia > 200 cells/µL had a sensitivity of 90% in predicting acute airway obstruction. The Characterizing the Response to a Leukotriene Receptor Antagonist and an Inhaled Corticosteroid (CLIC) trial showed that exhaled NO also significantly correlated with peripheral blood eosinophils, IgE and plasma ECP but not urinary leukotriene E4 (uLTE4) in 144 children with mild to moderate asthma who were between 6 and 17 years of age Exhaled NO levels and eosinophil counts have shown a positive correlation with IgEs specific to house dust mite (Figure 14).32 Atopic children with mild intermittent asthma, sensitized to house dust mite species, Dermatophagoides pteronyssinus (Dp) or D. farinae (Df), showed positive correlations between serum levels of total, Dp-specific or Df-specific IgE with esosinophil counts and exhaled NO levels.
AERO003-Proof 04 7/4/05 5:37 pm Page 20 P < 0.0001 P < 0.0003 Eosinophils (absolute) (/cu. mm) P < 0.0001 P < 0.0824 uLTE4 (pg/mg creatinine) Figure 13. Scattergrams showing that exhaled NO significantly correlates with (a) peripheral blood eosinophils; (b)plasma eosinophilic cationic protein (ECP); and (c) IgE; but not with (d) urinary leukotriene E4 (uLTE4) measurement onlog scale185 Prominent neutrophilia has been documented in certain asthma cases.205–207 Results suggest the presence of a distinct subgroup of patients with the clinical signs of asthma but who have predominantly Dp (RAST classes) neutrophilic airway inflammation; their airway secretions do not contain eosinophils. They also do not respond to steroid therapy, unlike patients with typical eosinophilic airway inflammation.205,208,209 P < 0.001 There is increasing evidence that neutrophils may play a role in acute severe Df (RAST classes) asthma. High levels of neutrophils have been demonstrated in fatal asthma of Figure 14. There are significant correlations betweenexhaled NO levels and (a) house dust mite Dp-specific sudden onset.210 Neutrophil numbers and IgE levels; (b) Df-specific IgE levels (P < 0.01)32Dermatophagoides pteronyssinus = Dp, D. farinae = Df activation are also increased during AERO003-Proof 04 7/4/05 5:37 pm Page 21 exacerbations of asthma.211 Jatakanon and colleagues showed that neutrophils were increased in patients with severe asthma compared with healthy volunteers (P < 0.05) and patients with mild asthma (P < 0.05).212 Ramesh and colleagues showed that production of nitrite and L-citrulline by neutrophils increased significantly as the severity of asthma increased from mild to severe (P < 0.001).19 Peak expiratory flow among all asthmatics correlated negatively with nitrite and L-citrulline, and NO production by neutrophils was increased in bronchial asthma, suggesting an association between NO production and progressive airway narrowing. Further evidence of a link between NO levels and neutrophilic inflammation comes from the use of an animal model.213 Lipopolysaccharide inhalation was shown to increase exhaled NO and this increase correlated to increases in airway neutrophilia and iNOS expression in lung tissues.
In an early study by Lim et al. steroid treatment was associated with a significant reduction in epithelial and submucosal immunoreactivity scores for eosinophils in bronchial biopsy specimens (P < 0.001 and P < 0.005, respectively), and significant reductions in the exhaled NO concentration (P < 0.001).214 A further study by the same group found a significant correlation between bronchial mucosal eosinophils and lung function (r = 0.43, P < 0.05), and significantly lower levels of exhaled NO in patients treated with inhaled steroids (P < 0.05).215 There was no direct correlation between mucosal eosinophils and exhaled NO Two more recent studies have provided strong evidence that exhaled NO reflects airway inflammation. van den Toorn and coworkers assessed the quantity of major basic protein in bronchial biopsies of patients with asthma and individuals in clinical remission.178 Significant correlations between major basic protein density and exhaled NO levels occurred in both groups (Figure 15).
Evidence of airway remodelling was also found in the patients in remission, even density epithelium though the median duration of remission Payne and colleagues examined children with difficult asthma and showed Figure 15. Exhaled NO levels correlate with major basicprotein density in bronchial biopsies from asthma patients a significant correlation between exhaled and individuals in remission from asthma178 AERO003-Proof 04 7/4/05 5:37 pm Page 22 NO levels and eosinophil scores in biopsies (r = 0.54, P = 0.03).179 In those Evidence of adherenceAdherence unkown patients with evidence of adherence to prednisolone, a NO level of less than 7 ppb (at a flow rate of 200–280 mL/s) was associated with an eosinophilic score in the non-asthmatic range, whereas all patients with persistent symptoms and eosinophil counts above the non-asthma range had NO levels above 7 ppb (Figure 16). However, in another study the Figure 16. Exhaled NO levels correlate with biopsyexaminations in children with difficult asthma179 same group did not find any correlation between NO levels and inflammatory cells in biopsies from children with difficult asthma.216 Clearly, further studies in this special group are required.
Young et al. studied the production of exhaled NO and eosinophil count, and the concentration of the chemokine eotaxin in BAL fluid of cynomolgus monkeys following an antigenic challenge.217 The concentration of NO in exhaled air doubled 24 h after antigen exposure (P < 0.05). Maximum levels of BAL eotaxin developed by 6 h after the challenge and were maintained at 24 h. There was also a dramatic increase in the BAL eosinophil count that was significant at 6 h and 24 h after the challenge. The changes in the BAL fluid thus follow a similar time course to the increase in NO production, despite the fact that there is no evidence that these events are causally related. A study by Lim et al. confirms a correlation between exhaled NO and inflammatory markers in BAL fluid in humans.215 Both exhaled NO and the percentage of BAL eosinophils were reduced by budesonide treatment, although only the decrease in NO was statistically significant. However, the reductions in exhaled NO and percentage of BAL eosinophils showed a statistically 1 – Specificity significant correlation (P < 0.05). In a study by Warke et al. including 71 Figure 17. Receiver-operated characteristic curve for thepresence of airway inflammation; exhaled NO > 17 ppb children with atopic asthma, atopic non- predicts inflammation with a sensitivity of 81%180 AERO003-Proof 04 7/4/05 5:37 pm Page 23 asthmatics and non-atopic normal controls, there was a significant correlation between the percentage of eosinophils in BAL fluid and exhaled NO.180 A BAL eosinophilic value of 0.86% represents the 95% confidence interval (CI) for the 95th percentile in normal children.
Using this as a cut-off value for airway inflammation, exhaled NO levels greater than 17 ppb (at 50 mL/s) predict airway inflammation with a sensitivity of 81% and a specificity of 80% (Figure 17). The authors concluded that exhaled NO measurement is a useful method of indirectly assessing eosinophilic airway inflammation in asthmatic children.
Pulmonary function tests represent the standard method for assessing asthma, although it is increasingly recognized that such tests do not reflect airway inflammation. Stirling and co- workers found no correlation between exhaled NO levels and lung function tests.218 These findings were also confirmed by Langley et al. who conducted a cross-sectional, hospital- based study of 392 patients with varied asthma severity, and found there was no correlation between exhaled NO and FEV .187 Furthermore, only a weak correlation between FEV and NO levels was reported by Sippel et al.219 Piacentini and colleagues have reported a study in which NO levels were monitored in a group of patients with atopic asthma who were placed in an Alpine home away from their allergens.220 NO levels fell during the 3 months in the Alpine home and remained stable even when glucocorticoids were withdrawn. Three weeks after returning to their usual homes, patients exhaled NO levels increased. In comparison, spirometry results continued to improve after the patients returned home (Figure 18), suggesting that such measurements respond slowly. Overall, most studies indicate that there is little correlation between Inhaled steroid withdrawal exhaled NO levels and pulmonary function tests in patients with asthma.
Furthermore, it appears that exhaled NO may respond more rapidly than Figure 18. Exhaled NO levels respond faster than spirometry to changes in allergen spirometry results to changes affecting airwayinflammation. FEV = Forced expiratory volume in exposure, thus making it a more 1 second; T0 = before admission to residential home; T1 = 2 weeks after T0; T2 = 3 months at Alpine home; sensitive marker of disease state.220,221 T3 = 2 weeks after return to normal home220 AERO003-Proof 04 7/4/05 5:37 pm Page 24 Exhaled NO in Asthma: Relationship to
Short- and long-acting β -agonists do not appear to affect the levels of exhaled NO in patients with asthma.222–225 Although one study has shown albuterol to have a simultaneous affect on the exhaled NO value. Within 20 minutes of albuterol treatment the NO value increased by 10–20%, but this effect was transient.62 NO and Corticosteroid Treatment
Inhaled corticosteroids have a marked effect on exhaled NO in keeping with their anti- inflammatory properties. The concomitant effect of inhaled steroid on markers of airway inflammation and exhaled NO has been examined in many studies. Lim and co-workers used a 12-week, double-blind, crossover design to compare the effects of inhaled budesonide with placebo.214 Each patient underwent 4-week periods of treatment with budesonide and placebo, separated by a 4- week washout period. Treatment with budesonide was associated with a significant drop in exhaled NO in parallel with improvements in other markers of inflammation (PC20methacholine, sputum eosinophils, and bronchial biopsy appearances). In this study, the improved indices of inflammation were matched by improvements in FEV , indicating reduced airway obstruction and a clinical improvement.
In line with these findings, van Rensen et al. reported a fall in exhaled NO with a simultaneous reduction in bronchial hyper-responsiveness (PC histamine) and sputum eosinophils during treatment with inhaled steroids.175 After the collection of baseline data, patients were treated with twice-daily inhaled steroids, or placebo, for 4 weeks, with further measurements taken at weeks 2 and 4. The final set of measurements was taken 2 weeks after the end of the treatment period. Markers of airway inflammation, including exhaled NO, improved significantly during the treatment period in the group receiving corticosteroids. Two weeks after the cessation of treatment, there was significant deterioration, compared with the values obtained at week 4. The level of exhaled NO therefore reflected the efficacy of anti-inflammatory Figure 19. Mean levels of exhaled NO in patients atbaseline, 2 weeks, 4 weeks, and after washout in patients therapy (Figure 19).
treated with steroids and placebo175 AERO003-Proof 04 7/4/05 5:37 pm Page 25 Aziz et al. assessed the effect of seven treatment regimens (placebo, formoterol [12 µg and 24 µg], budesonide [400 µg and 800 µg], and a combination of both [12 µg, 400 µg; 24 µg, 800 µg]), on exhaled NO in 15 patients with atopic asthma.224 Significant reductions were observed during treatment with regimens containing budesonide when compared with placebo. For example, mean exhaled NO levels after 2 weeks of budesonide 400 µg and 800 µg treatments were 8.3 ppb and 7.4 ppb at a flow of 83 mL/s. The corresponding value after placebo was 15.9 ppb.
A dose-dependent response of exhaled NO levels to corticosteroid treatment has been reported by Kharitonov et al.226,227 In this study, patients with asthma were treated with placebo, budesonide 100 µg or budesonide 400 µg. NO levels in the active treatment arms were decreased after Exhaled NO (% change) –40 just 3 days, with the largest decrease occurring in the group receiving the higher dose (Figure 20).227 Silkoff et al. also demonstrated an Figure 20. Exhaled NO levels in patients with asthma inverse relationship between inhaled BDP decreased in a dose-dependent manner duringcorticosteroid treatment with budesonide (BUD) 100 µg dose (0–800 µg/day) and the level of (closed squares) or budesonide 400 µg (closed circles).
Significant difference from placebo (open circles): exhaled NO (Figure 21).228 Exhaled NO was more effective for separating doses of BDP than FEV or PC . Furthermore, the fall in exhaled NO levels for a specific dose of BDP was highly reproducible.
Exhaled NO > 100 ppbAll patientsExhaled NO 60–100 ppb A study by Jones et al. confirms this dose relationship.229 Following withdrawal of inhaled BDP, 65 patients went though a double-blind, parallel group, and placebo- controlled trial of 50, 100, 200 or 500 µg BDP/day for 8 weeks. The relationship between the dose of BDP and change in exhaled NO was linear at 1 week and at the end of the study. The authors concluded that exhaled NO might be useful in guiding dipropionate (µg) dose adjustments of inhaled corticosteroids Figure 21. The response of exhaled NO to different doses in patients with persistent asthma.
of beclomethasone dipropionate228 AERO003-Proof 04 7/4/05 5:37 pm Page 26 A randomized, double-blind, placebo-controlled, crossover study with 4-week washout periods involving 18 adults with asthma was carried out to investigate changes in exhaled NO levels after inhaled and oral anti-inflammatory therapy.230 A significant difference in mean exhaled NO levels was found (P < 0.01) before and after low-dose inhaled fluticasone propionate (FP; 44 µg): 34 ± 7 ppb vs. 13 ± 3 ppb, respectively. There was also a significant improvement in FEV % (from 75 ± 3 to 85 ± 3; P < 0.05). No significant reduction was found in exhaled NO levels with low-dose oral zafirlukast (20 mg) for 4 weeks.
The above studies were performed in adult patients, but corticosteroids, both inhaled and systemically administered, have also been demonstrated to be effective in reducing the amounts of exhaled NO in children with acute and stable asthma (Figure 22).75,231–233 For example, Carrà et al. showed that exhaled NO levels were reduced by over 40% when children with stable asthma were treated with budesonide (400 or 600 mg/day) for 6 weeks.234 Covar et al. also showed that budesonide therapy was more effective than nedocromil in reducing exhaled NO Figure 22. Individual (open circles) and group mean(closed circles) exhaled NO values before and after a levels.176 Budesonide-treated children had 5-day course of oral prednisolone therapy in children with significantly lower median exhaled NO asthma. Vertical bars denote the SEM. Both before andafter steroid therapy, exhaled NO values for children with levels and ECP levels than those receiving asthma were significantly elevated (P < 0.001) comparedwith those of control children75 placebo (21.5 ppb [95% CI 13.2, 84.4] vs.
62.5 ppb [95% CI 26.2, 115.0], P < 0.01; 17.4 mg/dL [95% CI 10.1, 24.3] vs. 24.0 mg/dL [95% CI 15.4, 33.9], P = 0.5, respectively). Exhaled NO in children with asthma has also been shown to respond to a dipropionate aerosol.235 Another study demonstrated that although exhaled NO is within the normal range in most asthmatic children on a moderate dose of inhaled corticosteroids (budesonide), exhaled NO exhibited a heterogeneous response to corticosteroids and levels remained high in Figure 23. Change in mean exhaled NO levels after a subgroup of clinically well controlled 2 weeks of treatment in adults and children withuncontrolled asthma.237 Exhaled NO assessed at children with asthma.236 50 mL/s using NIOX® AERO003-Proof 04 7/4/05 5:37 pm Page 27 In a study involving both adults and children with uncontrolled asthma, 2 weeks of corticosteroid treatment resulted in a mean percentage reduction of 50.5% (Figure 23).237 Notably, all patients who did not show a reduction in their NO levels of Exhaled NO (% change) over 20% did not report a reduction in Corticosteroids inhibit the induction of Figure 24. Effect of inhaled budesonide on exhaled NO iNOS both in vitro and in vivo, and the fall in patients with mild asthma. There was a significantreduction after inhaled budesonide (closed circles) in exhaled NO appears to be a reflection of 1600 µg daily, but not after matched placebo (open this.238 A reduction in exhaled NO is circles). Mean values (± SEM) for 11 patients are shown.
Significant difference from baseline: *P < 0.05; **P < 0.02, clearly demonstrable within 1 week of ***P < 0.01. Differences between the budesonide andplacebo treatment periods were also significant at 7, 14 starting treatment (Figure 24).239 and 21 days ( P < 0.05)239 In acute exacerbations of asthma, the effect of steroid treatment can be seen even faster.
Massaro et al. demonstrated a fall in exhaled NO 48 h after starting corticosteroid treatment,240 and Baraldi et al. found a mean reduction in the level of exhaled NO of 46% after 5 days of treatment with oral prednisone,75 which was confirmed in a later study by the same group.241 The use of nebulized budesonide in acute asthma has been shown to significantly reduce NO levels just 6 h after treatment.242 Interestingly, the reduction in NO in this study correlated significantly with changes in peak expiratory flow (PEF).
As may be expected, it has been shown that treatment of asthma with the corticosteroid pro-drug, ciclesonide, is associated with a decrease in exhaled NO.243 Asthma management guidelines advocate the addition of a long-acting β -agonist to inhaled corticosteroids as an alternative to increasing the dose of the latter.244 It is thought that long-acting β -agonists may exert a facilitatory effect on inhaled corticosteroids permitting a lower corticosteroid dose. In light of this, studies have investigated the effects of combination therapy on exhaled NO levels.81,245,246 Currie et al. evaluated the anti- inflammatory activity of fluticasone plus salmeterol in combination versus a double dose of fluticasone.81 Fifteen people with mild-to-moderate asthma (mean FEV 80% predicted) that was uncontrolled on inhaled corticosteroids were randomized in a single-blind crossover study to receive 2 weeks each of fluticasone 250 µg plus salmeterol 50 µg in combination and fluticasone 500 µg. Both fluticasone and fluticasone plus salmeterol conferred a significant (P < 0.05) fall in exhaled NO from baseline. However, between treatments, the reduction was significantly (P < 0.05) greater with fluticasone treatment AERO003-Proof 04 7/4/05 5:37 pm Page 28 alone (Figure 25). Buchvald and Bisgaard FP = fluticasone 500 µg FP + SM = flutiocasone 250 µg + compared exhaled NO levels after salmeterol 50 µg salmeterol or montelukast add-on therapy in 22 asthmatic children receiving regular maintenance treatment with budesonide 400 mg daily.246 Exhaled NO levels were significantly higher after salmeterol add- Exhaled NO (ppb) on treatment compared with both placebo (P = 0.003) and montelukast (P = 0.002) add-on treatment. Furthermore, salmeterol also improved lung function (FEV ) significantly compared with Figure 25. Both fluticasone (500 µg) and FP (250 µg) + salmeterol (50 µg) conferred a significant (*P < 0.05) placebo and non-significantly compared fall in exhaled NO levels from baseline; this reduction wassignificantly lower (†P < 0.05) with fluticasone than with with montelukast.
fluticasone + salmeterol75 NO and Anti-Leukotriene Treatment
Cysteinyl leukotrienes are produced and released by inflammatory cells in the airways of asthmatic patients and are important mediators of asthma. Leukotriene pathway modifiers have been shown to improve asthma control and may have a more prolonged action than corticosteroids.247 In a study of children with mild-to-moderate stable chronic asthma, not requiring maintenance steroids, Bratton et al. have demonstrated that the receptor antagonist montelukast sodium significantly reduced exhaled NO by approximately 33%.247 The reduction in exhaled NO was not accompanied by any significant change in tests of pulmonary function, suggesting that exhaled NO is a more sensitive measure of inflammation than tests of lung function. These findings are supported by the results of a double-blind crossover study by Bisgaard et al.248 who compared a 2-week treatment with montelukast, 5 mg daily, with placebo. Montelukast sodium treatment was associated with a significant (20%) reduction in exhaled NO (Figure 26), and most of this effect (15% reduction) was evident within 2 days. Although there was a Montelukast Budesonide tendency towards improved lung function with montelukast treatment, this did not Figure 26. NO in exhaled air of asthmatic children isreduced by the leukotriene receptor antagonist reach statistical significance. AERO003-Proof 04 7/4/05 5:37 pm Page 29 One study has described more precisely the time course of changes in exhaled NO with montelukast therapy. Montelukast mg daily), administered in a randomized, double-blind crossover design over 2 weeks, significantly reduced the levels of exhaled NO from the first day of treatment with the maximal effect Exhaled NO change (ppb) occurring on the seventh day (median change, 22%; Figure 27).98 Furthermore, the levels of exhaled NO remained lower in comparison to baseline during the Figure 27 Montelukast treatment resulted in a significantreduction in exhaled NO levels from day 1 of treatment. D washout period.
= day of treatment; W = day of washout98 Ghiro and colleagues studied the effect of adding montelukast to inhaled corticosteroid treatment in children. After 3 weeks there was a significant reduction in exhaled NO values in the group treated with both montelukast and inhaled steroids compared with the group remaining on inhaled corticosteroids only. After withdrawal of the montelukast therapy the NO values rose to baseline levels again.249 This suggests an anti-inflammatory effect of montelukast, additive to that of inhaled corticosteroids. Lee et al. also reported a lowering of NO levels when montelukast was added to a inhaled corticosteroid treatment, in this case after only 1 week of treatment.250 Further support comes from Lipworth and colleagues, who examined the effects of adding the leukotriene antagonist, zafirlukast, or a β -agonist, to corticosteroid treatment in 24 asthmatic patients.251 In this crossover study, addition of zafirlukast for 1 week resulted in a significant decrease in exhaled NO. In contrast, no significant decrease was seen after the addition of the β -agonist to corticosteroid treatment. Whelan et al. found montelukast had no significant effect on exhaled NO levels.67 However, when patients were stratified according to the genotype of the leukotriene C4 (LTC4) synthase A C polymorphism, montelukast significantly reduced the slope of the percentage change in exhaled NO levels compared with time curve in heterozygotes, suggesting that this subgroup responded better to montelukast therapy with respect to exhaled NO levels.
The lack of an effect of antileukotrienes on NO levels that has sometimes been reported may be due to differences in the pathology behind increased NO levels and increased leukotrienes. It has been shown that exhaled leukotriene levels do not respond to corticosteroids to the extent that NO levels do, possibly indicating weak pathophysiological AERO003-Proof 04 7/4/05 5:37 pm Page 30 Leukotriene antagonist therapy in patients with asthma may also improve exercise tolerance. Montelukast was shown to reduce exhaled NO levels in response to exercise in people with mild to moderate asthma, but had little effect on bronchial hyper-responsiveness to methacholine and adenosine challenges.253 NO and Other Anti-Inflammatory Treatment
Reductions in exhaled NO, accompanied by improved indices of lung function, were observed by Borish et al. in a study of asthmatic patients treated with the soluble interleukin- 4 receptor (IL4R).254 Patients taking placebo or low-dose IL4R showed deterioration in asthma symptoms and increased exhaled NO after abrupt steroid withdrawal, but patients taking high-dose IL4R had significantly fewer asthma symptoms and lower exhaled NO concentrations. Objective tests of lung function, including FEV and forced expiratory flow ), showed less deterioration with high-dose treatment, and patients in this group also needed less rescue medication with inhaled β -agonists. Effective anti- inflammatory treatment thus reduced clinical and spirometric indicators of asthma severity, and reduced exhaled NO mirrored this.
Omalizumab is a monoclonal antibody to IgE that is used in the treatment of moderate-to- severe atopic asthma. One preliminary study suggests that omalizumab reduces exhaled NO levels to a similar extent to inhaled corticosteroids, indicating an anti-inflammatory affect for Correlation with Disease Severity and Control
Although exhaled NO is reduced by inhaled corticosteroids, it is not totally suppressed and continues to show a correlation with the severity of disease. Stirling et al. found that patients with asthma that was difficult to control (i.e. requiring high doses of inhaled or oral steroids) had exhaled NO levels lower than the levels in steroid-naïve asthmatics, but significantly higher than those in normal controls.218 Exhaled NO levels correlated closely with clinical markers of disease control (symptom frequency and the need for rescue β -agonist use), although not with tests of lung function. Similarly, Artlich et al. found that children with recent symptoms of bronchial obstruction had high levels of exhaled NO, even if steroids were included in their medication.256 These findings have been confirmed by other groups suggesting that exhaled NO levels may serve a useful role in monitoring response to medication changes and assessing patient compliance.176,257 Meyts and colleagues compared exhaled NO levels with the clinical assessment of asthma control in 73 children with asthma aged 5–18 years.257 They showed that exhaled NO levels were higher in patients in whom asthma control was insufficient (Figure 28), whereas levels were not significantly different between children in whom asthma was well controlled and in those whose control was deemed acceptable. In another study, AERO003-Proof 04 7/4/05 5:37 pm Page 31 P = 0.01 Figure 28. Significantly higher exhaled NO levels were Figure 29. Exhaled NO levels (at 50 mL/s) increase as measured in patients with insufficient asthma control asthma severity increases258 than in patients with good asthma control and in patientswith acceptable asthma control257 NO levels correlated significantly to the severity of asthma based on the National Asthma Education and Prevention Program (NAEPP) guidelines (Figure 29).258 The effect of corticosteroids on exhaled NO is dose related, as reported by Kharitonov et al. in a study performed on adult asthmatics maintained on twice-daily inhaled budesonide.226,238 After a 200 µg reduction in the dose of budesonide, the exhaled NO concentrations rose. Although there were no significant changes in lung function or daytime asthma symptoms, there was a significant increase in nocturnal asthma symptoms. When the dose of budesonide was increased to 200 µg more than the usual maintenance dose, the level of exhaled NO fell, and this was associated with a reduction in the diurnal variability of PEF and a reduction in nocturnal symptoms. Thus, the level of exhaled NO is a marker of disease activity and may be more sensitive than either lung function tests or clinical symptoms. Exhaled NO levels have also been assessed in children with stable asthma whose doses of corticosteroids were adjusted according to National Institutes of Health (NIH) guidelines.259 Notably, NO levels were higher in the children who required an increase in dose than in similar children who did not need a dose increase (Figure 30). Thus, exhaled NO levels correlated (although only weakly) with the decision on treatment, even though they were not used as a guide for that decision.
No statistical correlation was found between exhaled NO levels and the Figure 30. Exhaled NO levels correlate with treatment disease severity in this study. AERO003-Proof 04 7/4/05 5:37 pm Page 32 In another similar study, exhaled NO levels were also found to be higher in patients whose corticosteroid dose was increased after examination by a paediatric pulmonologist or an allergist, compared with those whose dose was unchanged.260 In addition, NO levels correlated with the change in FEV . The authors suggested that NO levels could be a clinically useful measure of asthma severity.
Sippel et al. examined 100 patients (age range 7–80 years) with asthma, using a questionnaire, spirometry and exhaled NO.219 Exhaled NO levels correlated with asthma symptoms during the previous 2 weeks, dyspnoea score, daily use of rescue medicines, and the reversibility of airflow obstruction. There was, however, no correlation between exhaled NO levels and history of respiratory failure, healthcare use, fixed airflow obstruction or a validated asthma score. Nevertheless, the authors concluded that monitoring of exhaled NO might be useful in outpatient management as a means of determining asthma control.
In a study by Szefler and colleagues, the response to anti-inflammatory treatment was found to be correlated with the exhaled NO level before treatment. A good (> 15%) FEV1response was found to be associated with high exhaled NO (17.4 ppb), compared with a poor (> 5%) response, which was associated with lower exhaled NO levels (11.1 ppb).261 In another study by Szefler et al., children with high NO levels, high levels of other inflammatory markers, and low pulmonary function at baseline were more likely to respond to fluticasone than other children with asthma.262 Thus, the authors conclude that NO levels and other markers can be used to indicate the requirement for inhaled corticosteroids.
Reid et al. found that exhaled NO levels reflected clinical activity in asthmatic patients treated with inhaled corticosteroid.263 Furthermore, exhaled NO levels also correlated with blood eosinophils and airway hyper-responsiveness but not eosinophilic airway Quality of Life
It is increasingly evident that exhaled NO measurements reflect the underlying airway inflammation in asthma. Patients' general wellbeing has been found to correlate to their airway inflammation status. In a longitudinal study, Grönke et al. studied patients with severe asthma who had NO levels, sputum eosinophils, lung function and quality of life (Juniper scale) assessed over 18 months.264 Results showed a significant negative correlation between NO levels and quality of life measurements (r = –0.61 at 18 months; P < 0.05), whereas there was no correlation between lung function and quality of life. Sputum eosinophils also correlated with quality of life, but less strongly than NO. AERO003-Proof 04 7/4/05 5:37 pm Page 33 Clinical Use of Exhaled NO in Asthma
Measurement of exhaled NO provides a useful method of differentiating asthma from other conditions. The diagnostic value of NO measurements and the ability of the method to differentiate between healthy subjects (with airway symptoms) and patients with true asthma were analysed in an early study by Dupont et al.265 The sensitivity and specificity of the method is dependent on the selection of an appropriate cut-off point. In the study, 150 consecutive patients (79 females, 71 males, mean age 41 years, non-smokers, steroid- naïve) with symptoms suggestive of obstructive airways disease were examined. Of these, 108 were diagnosed as asthmatic on the basis of significant airway reversibility and airway hyper-responsiveness to histamine. At a cut-off value of > 12 ppb (flow rate not stated), a sensitivity of 81% and a specificity of 80% were seen. A further study by the same group in 240 consecutive, non-smoking, steroid-naïve patients found that a cut-off point for exhaled NO of 16 ppb (at a flow rate of 200 mL/s), gave a specificity for the diagnosis of asthma of 90% and a positive predictive value of > 90%.266 With a cut-off of 20 ppb (flow rate 200 mL/s), a specificity of 100% was seen.
Henriksen and co-workers investigated the use of exhaled NO levels alone (using a cut-off value of 8 ppb at 250 mL/s) or in combination with airway hyper-responsiveness tests to diagnose asthma in a large population survey (n = 8571).29 The study showed that 52% of those diagnosed with asthma had NO levels at or above the cut-off value, whereas 80% of those who were thought to be healthy had NO levels below 8 ppb. The authors suggested that combining this NO cut-off level with airway hyper-responsiveness to methacholine (< 2 mg causing a 20% fall in FEV as cut-off value) would allow the diagnosis of asthma with a high Chatkin et al. have investigated the value of NO in the assessment of chronic cough.93 Using 30 ppb (at a flow of 45 mL/s) as the cut-off point for exhaled NO gave a sensitivity and specificity of 75% and 87%, respectively, for a diagnosis of asthma. In a non-smoking adult population of asthmatics and healthy controls, an exhaled NO measurement of 30 ppb at a flow rate of 42 mL/s was both sensitive and specific for a diagnosis of asthma.267 The positive predictive value was 72%, and the negative predictive value was 71%. If used as a screening test in the general population, the authors calculated that exhaled NO measurements of less than 30 ppb would have a negative predictive value of 98% (Figure 31). AERO003-Proof 04 7/4/05 5:37 pm Page 34 In a study of 71 children undergoing No discriminationExhaled NO elective surgery, Warke and colleagues found that exhaled NO measurements greater than 17 ppb were both a highly sensitive (81%) and highly specific (80%) means of predicting airway inflammation.180 (See Section III Exhaled NO: Correlation with Known Inflammatory Sensitivity (true positives) Markers, F. Bronchoalveolar Lavage.) 1 - Specificity (false positives) Another study by Malmberg et al.90 showed that airway inflammation was Figure 31. Receiver-operated characteristic (ROC) curveshowing the value of various exhaled NO concentrations present at the early stages of asthma, even in the diagnosis of asthma267 in pre-school children (3.8–7.5 years old).
Exhaled NO was superior to baseline lung function measures or indices of bronchodilator responsiveness, assessed by the oscillometric technique for identifying pre-school children with predominantly atopic probable asthma (Figure 32). The optimum cut-off value for exhaled NO was 9.7 ppb (flow rate 50 mL/s) giving a sensitivity of 86% and specificity of 92%. Also, exhaled NO had a high negative predictive value of 95%.
Asthma may be defined either as wheeze within the previous 12 months (current wheeze), doctor-diagnosed asthma, or current wheeze plus confirmed airway hyper-responsiveness.
Gender may also affect how asthma is diagnosed. Henriksen et al. found that there is a risk of underestimating the prevalence of asthma, especially among girls, when asthma is defined as doctor-diagnosed asthma (exhaled NO levels were measured at a rate of 250 Exhaled NOBaseline lung function A comparison of exhaled NO levels and sputum cell counts with a range of clinical tests normally recommended by international guidelines to confirm the diagnosis of asthma was recently carried out by Smith and colleagues. In this study, asthma was defined by a positive response to a bronchodilating agent or a positive hyper-responsiveness test, in accordance with ATS guidelines. Sensitivities for each Figure 32. ROC analysis showed exhaled NO had the bestdiscriminative capacity (area under the ROC curve: 0.91, of the conventional tests (peak flow 95% CI: 0.83–0.96) followed by baseline lung indices90 AERO003-Proof 04 7/4/05 5:37 pm Page 35 measurements and spirometry) following a steroid trial were lower (0–47%) than for exhaled NO (88%) and sputum eosinophils (86%).170 Both exhaled NO and sputum eosinophils provided significantly higher degrees of diagnostic accuracy than tests based on lung function (Figure 33). For exhaled NO, the optimum cut-off point for diagnosing asthma, based on calculating the predictive accuracy for a range of different exhaled NO levels, was 20 ppb (at a flow rate of 50 mL/s). The authors concluded that exhaled NO measurements Figure 33. Exhaled NO (solid line) was more accurate(sensitivity 88% at a cut off of 20 ppb) than lung function are superior to the conventional tests that tests (dotted line) for the diagnosis of asthma170 are recommended by international guidelines in diagnosing asthma.
Response to Anti-Inflammatory Treatment
One of the most useful features of exhaled NO is the response to anti-inflammatory treatment, enabling physicians to monitor the effect of treatment objectively. There are now substantial data showing that corticosteroids reduce exhaled NO in asthma, as discussed above. The response of exhaled NO to anti-inflammatory treatment is both rapid269 and dose-dependent.226–229 Thus, exhaled NO measurements have the potential to be used in monitoring the effects of such treatment.
Little et al. have shown that baseline NO levels correlate with the percentage improvement in FEV obtained after steroid and bronchodilator treatment.270 In addition, Szefler and coworkers have shown that high baseline NO levels are a predictor of response to inhaled corticosteroid treatment.262 Reid et al. showed that exhaled NO levels, despite being within the normal range in inhaled corticosteroid treated patients with persistent asthma, remained significantly related to airway hyper-responsiveness, blood eosinophils and clinical markers of disease severity.263 Although treatment with inhaled corticosteroids reduces exhaled NO, it does not totally suppress it and therefore a correlation with the severity of disease is still evident.231,256 (See Section IV Exhaled NO in Asthma: Relationship to Anti-Inflammatory Treatment, E. Correlation with Disease Severity and Control.) AERO003-Proof 04 7/4/05 5:37 pm Page 36 Exhaled NO measurement may be of great value in monitoring asthma and assessing the response to treatment, i.e. mainly in tracking the degree of inflammation and airway reactivity within individual patients.271 Monitoring Compliance with Anti-Inflammatory Treatment
Failure to comply with inhaled corticosteroid therapy may be a larger problem than is often recognized. A study by Milgrom et al. compared data on inhaler use from electronic inhaler monitors with data collected by asthmatic children using diary cards.272 More than 90% of children exaggerated their use of inhaled steroids. According to the diary cards, median use of inhalers was 95.4% of the prescribed dosage, whereas the median actual use was 58.4%.
Furthermore, the least compliant patients were the most likely to have acute exacerbations of disease. Compliance is therefore a major issue in the management of childhood asthma. Beck-Ripp et al. studied 34 patients with asthma treated with budesonide for 4 weeks, followed by a washout period and then randomized to budesonide or no budesonide for 8 weeks.273 Compliance was measured by assessing the number of doses remaining in the delivery device at each visit. NO levels were significantly Reduction in exhaled NO (%) reduced during the run-in period but increased during the washout period. As Compliance with budesonide might be expected, NO decreased again in (% of prescribed) those patients randomized to budesonide, Figure 34. Relationship between exhaled NO andcompliance with inhaled steroids273 whereas NO levels where unchanged in patients not receiving budesonide. A very good correlation was found between NO levels and compliance (r2 = 0.586; P = 0.0003) (Figure 34). A similar correlation was found by Delgado-Corcoran and coworkers (r2 = 0.56), who demonstrated the large difference in NO levels between patients who exhibited poor compliance (≤ 49% of prescribed regimen) and those who showed good compliance (>75% of prescribed regimen) (Figure 35).258 Exhaled NO may thus be a valuable parameter to Figure 35. Exhaled NO levels (at 50 mL/s) in patients withasthma who demonstrated poor, moderate or good monitor adherence to steroid treatment.
compliance with corticosteroid treatment258 AERO003-Proof 04 7/4/05 5:37 pm Page 37 Detection of Steroid Unresponsiveness
Although anti-inflammatory treatment usually reduces NO, some patients continue to have persistently elevated levels of exhaled NO, despite corticosteroid treatment. Possible reasons suggested for such findings include poor inhalation technique, inadequate corticosteroid dosage, peripheral or overwhelming inflammatory activity, and non-compliance with treatment.254 There may also be a small number of patients, especially those with difficult or severe asthma, who are unresponsive to steroid treatment.218,274 With regard to steroid-resistant asthma, insights have been reported by Payne et al.274 In this study, NO levels were assessed before and after oral prednisolone treatment in children with difficult asthma. In some of these patients, NO levels were high at baseline and remained high following treatment, and this group continued to have persistent symptoms.
Notably, some patients had normal levels of NO at baseline that remained normal during treatment. However, several of these patients still had persistent symptoms after treatment.
The authors concluded that NO levels may be used to identify different subsets of patients with difficult asthma. Those with high levels of NO, which remain high, may require a higher dose or a different type of anti-inflammatory treatment. In contrast, patients with normal NO levels that show no symptomatic response to anti-inflammatory treatment may have little or no eosinophilic inflammation, and thus, continued anti-inflammatory treatment may be Prediction of Loss of Control of Asthma
Prevention of exacerbations is an important goal in the management of asthma, and prediction of exacerbations before the onset of clinical symptoms and airway obstruction could be of considerable value. One prospective study induced mild exacerbations of asthma by reducing the dose of steroids in stable asthmatic patients maintained on inhaled corticosteroids.197 Seven of 15 patients developed mild exacerbations of asthma over the course of the 8-week study. At baseline, the only difference between those who did and those who did not develop an exacerbation was a higher baseline sputum eosinophil count. Exhaled NO increased during the study in those patients who developed exacerbations. The increases in sputum eosinophils and exhaled NO were correlated with decreases in airway function (morning PEF and FEV ). Multiple regression analysis suggested that the change in sputum eosinophils is a potentially useful marker in predicting loss of asthma control as reflected by loss of airway function (Figure 36).
AERO003-Proof 04 7/4/05 5:37 pm Page 38 Figure 36. Exhaled NO levels and symptoms in asthma patients with mild exacerbations after glucocorticoid dosereduction.197 Compelling evidence comes from Jones and co-workers, who examined the ability of exhaled NO, sputum eosinophil and hyper-responsiveness measurements to predict loss of control.188 In the study, 78 patients with mild to moderate asthma stopped their corticosteroid treatment and were followed for 6 weeks. Exhaled NO greater than 15 ppb (at a flow of 250 mL/s) at baseline had a positive predictive value of 88%, but the sensitivity was low (25%). However, NO values at the visit prior to loss of control and changes in NO from baseline to this visit were shown to have good positive predictive values and better sensitivities (Table 4). In addition, NO levels were as good at predicting loss of asthma control as the other markers. The authors concluded that because of simplicity of use, exhaled NO was the preferred marker. They also suggest that lower flow rates (i.e. 50 mL/s) may have allowed greater distinction between those who developed exacerbations and those who did not, leading to higher sensitivities, specificities and positive predictive values than those Table 4. Exhaled NO levels predict loss of control of asthma.188
Sensitivity
NO at visit prior to loss 0.83 (0.67, 0.94) 0.50 (0.37, 0.63) 0.65 (0.38, 0.86) of control > 15 ppbChange in NO from 0.83 (0.67, 0.94) 0.50 (0.37, 0.63) 0.65 (0.38, 0.86) baseline to visit prior to loss of control > 60%Percentage eosinophils 0.80 (0.52, 0.96) 0.21 (0.12, 0.34) 0.80 (0.52, 0.96) at baseline > 4%Saline PD at baseline 0.77 (0.60, 0.90) 0.53 (0.38, 0.67) 0.50 (0.25, 0.75) AERO003-Proof 04 7/4/05 5:37 pm Page 39 Similar results were shown in a study of 37 well-controlled asthmatic patients by Prieto et al., though bronchoconstriction in response to AMP or exhaled NO levels alone was not a predictor for failure in corticosteroid reduction.194 In another study, exhaled NO was significantly higher in patients who subsequently had an exacerbation within 2 weeks of the measurement (29.7 ± 14.5 vs. 12.9 ± 5.2).275 Logistic regression showed that exhaled NO was the only significant predictor of exacerbation (Table 5) Table 5. Most significant assessments predicting asthma exacerbations.275
An early study on 10 children with asthma presenting to the emergency room during acute asthmatic exacerbations showed that the mean exhaled NO levels prior to and after glucocorticoid treatment were 48 ± 8 ppb and 17 ± 1 ppb, respectively (P < 0.002).231 FEV % and PEF both improved after treatment. Results suggested that exhaled NO is a sensitive marker of acute asthma exacerbations in children.
Safe Withdrawal of Inhaled Corticosteroids
After withdrawal of inhaled corticosteroids, many children previously diagnosed with asthma do not develop airway hyper-responsiveness. One study investigated whether differences between children with and without airway hyper-responsiveness after withdrawal of inhaled corticosteroids were compatible with differences between transient and persistent wheezers found in other studies. Investigators found that hyper-responsive children had more atopic features (positive RAST, high IgE, eczema) and lower FEV values and soluble intercellular adhesion molecule-1 (ICAM1) levels than non-hyper-responsive children.276 Hyper-responsive children also had elevated exhaled NO levels and lower levels of lung function. Children with airway hyper-responsiveness after withdrawal of inhaled corticosteroids shared features with persistent wheezers as observed in epidemiological studies. Children without airway hyper-responsiveness after withdrawal of inhaled corticosteroids probably had transient wheeze.
An interesting study published in The Lancet shows that steering the anti-inflammatory dose according to the patient's inflammation status results in a healthier patient. Green et al.
treated two groups of patients, one by normalizing the sputum eosinophil level and the other AERO003-Proof 04 7/4/05 5:37 pm Page 40 according to present British guidelines.277 BTS management group Sputum management group Treatment based on the inflammatory marker resulted in fewer emergency room visits (1 compared to 6 of 37 patients, P = 0.047) and patients in the sputum management group had significantly fewer severe asthma exacerbations than patients managed by standard regimens (35 vs. 109; P = 0.01) (Figure 37). This study provides proof for the concept that monitoring inflammation and dose titration according to the inflammatory status can lead to real Figure 37. The sputum management group hadsignificantly fewer severe exacerbations compared clinical benefits.
with the BTS management group (35 vs. 109 totalexacerbations, respectively, P = 0.001)277 As the measurement of exhaled NO is a simpler and less time-consuming technique than the measurement of sputum eosinophils, it is more suitable for routine clinical practice and allows more frequent measurement of inflammation. Studies where exhaled NO is used as the inflammatory marker for dose-optimization have not yet been published.
Exhaled NO as an Early Marker of Asthma
Several publications have indicated that exhaled NO levels may be increased before asthma symptoms develop. Moody and co-workers examined NO levels and skin-prick tests in 64 asymptomatic Pacific Islanders – a racial group known to have a high risk of developing asthma.34 Individuals sensitive to house dust mites had high levels of exhaled NO, which correlated with the severity of their sensitivity. The authors concluded that raised NO levels in this population may represent subclinical airway inflammation.
de Kluijver and colleagues have shown that ‘silent' chronic allergen exposure can induce and maintain airway inflammation, which can be prevented with anti-inflammatory treatment.278 Repeated low-dose allergen exposure to house dust mites resulted in a significant increase in sputum eosinophils, ECP and exhaled NO, compared with the Asthma symptoms often decline during puberty and some patients experience clinical remission in early adulthood. However, a proportion of those who experience remission have relapses later in adult life. van den Toorn and colleagues have shown that adults in remission can have airway inflammation that correlates with NO levels.178 The authors speculated that this subclinical inflammation may be a risk factor for asthma relapse in later AERO003-Proof 04 7/4/05 5:37 pm Page 41 As well as being elevated in asthma, exhaled NO levels are also elevated, though to a lesser extent, in non-asthmatic rhinitis.45 This finding suggests that exhaled NO levels may help identify patients at risk of developing asthma.
The relationship between exhaled NO levels and subclinical airway inflammation has also been investigated in children. Franklin and colleagues measured NO levels in healthy children and the level of formaldehyde in their homes.279 Formaldehyde has been associated with adverse respiratory symptoms in both children and adults. NO levels were significantly higher in children living in homes with formaldehyde levels above 50 ppb than in those living in homes with less formaldehyde. Notably, there was no correlation between formaldehyde levels and spirometry results.
Although more studies are required, it is possible that exhaled NO levels may prove useful in identifying individuals at risk of developing asthma.
Exhaled NO in Epidemiology
The epidemiology of asthma is of high interest, as researchers seek insights into the reasons behind the general increase in the prevalence of the condition. Epidemiologists have generally relied on symptoms and lung function tests to gain information on factors that affect asthma risk, but to fully understand the natural history of asthma, information is needed on factors that affect airway inflammation. Exhaled NO, particularly with the advent of a portable device, now offers a convenient method of assessing airway inflammation in such studies. Saito and colleagues have tested the potential of exhaled NO in an epidemiologic study involving children.280 They found that exhaled NO was the best predictor of recurrent wheeze, when compared with IgE and pulmonary function tests. Given this and the convenience of the test, the authors suggest that exhaled NO is a valuable marker of inflammation that is suitable for epidemiological investigations.
One epidemiolgocial study in the Netherlands investigated the differences in allergic sensitization and NO levels between children of different ethnic origin.281 Exhaled NO levels were higher in children with allergic sensitization than in those without any sensitizations.
Children of Moroccan origin who were sensitized to indoor allergens had the highest levels of NO. A community study in Australia also showed that allergic sensitization was associated with increased NO in levels in schoolchildren.129 With the growing use of exhaled NO measurements in epidemiology, one can expect to see further insights into the factors that affect the risk of developing asthma. One potential risk factor that is of great interest is air pollution.
AERO003-Proof 04 7/4/05 5:37 pm Page 42 Air Pollution and NO Levels
Air pollution is known to affect the health of patients with asthma, but its role in the development of the condition is more controversial. A birth cohort study in Norway (the Environment and Childhood Asthma study) found no correlation between outdoor air pollution and the occurrence of bronchial obstruction in the first 2 years of life.282 However, no assessment of the effects of pollution on airway inflammation was made in the initial part of this study (a follow-up involving exhaled NO measurements is ongoing).
A number of studies have assessed exhaled NO in relation to air pollution. Steerenberg and coworkers found that exhaled NO levels in children were directly associated with environmental levels of black smoke, nitrogen dioxide, NO and particulate matter < 10 µm.283 Fischer et al. also reported that exhaled NO levels in children increased (by as much as 31%) with increasing air pollution levels.284 Notably, there was no association between changes in lung function and air pollution. Given this, and the fact that exhaled NO levels correlated with mild respiratory symptoms (e.g. sore throat, runny nose), the authors concluded that exhaled NO was a more suitable measure of the health effects of air pollution. Exhaled NO levels also increase in adults exposed to higher levels of air pollution.
Steerenberg and colleagues noted a 67–78% increase in exhaled NO on days with high levels of ambient NO.285 A significant increase in exhaled NO levels in association with a 17.7 µg/m3 increase in levels of particulate matter < 2.5 µm has been reported in elderly individuals.286 Importantly, the association between NO and particulate matter was stronger in patients with COPD.
All the studies described previously in this subsection have assessed response to environmental air pollution. However, a laboratory study failed to find significant changes in NO levels in response to increasing exposure to carbon ultrafine particles in both healthy individuals and patients with asthma.287 The reason for this apparently contrasting finding is not known, but could be due to the contrast between the relatively acute exposure in the laboratory setting and the chronic exposure in the epidemiological setting.
NO Levels and Occupational Health
Occupational asthma is a continuing problem, and exhaled NO is potentially useful for identifing workers at risk of developing asthma after exposure to occupation-related Lund et al. reported increased exhaled NO levels in 99 non-smoking aluminium pot-room workers compared with controls.289 Only 12 of the workers with high NO levels had asthma- like symptoms. In another study, exhaled NO was assessed in workers after challenge with AERO003-Proof 04 7/4/05 5:37 pm Page 43 4,4'-diphenylmethane diisocyanate, natural rubber latex or methacholine.288 Individuals with substance-specific IgE antibodies and a bronchial response to stimulants had a tendency to develop increased NO levels shortly after (22 h) the challenge.
Olin et al. have shown that exhaled NO levels are higher in pulp mill workers who have experienced ozone gassing incidents than in those who have not reported such incidents.190 The authors suggested that the high NO levels may have been due to chronic airway inflammation. In a follow-up study 3 years later, those workers who had the highest exposure to ozone had significantly higher NO levels than controls and an increased prevalence of adult-onset asthma.96 Sundblad et al. found that bronchial responsiveness and exhaled NO increased after exposure to a swine confinement facility.290 Exhaled NO levels have also been shown to increase (by 40%) in shoe and leather workers during their working day, probably as a result of exposure to organic solvents.291 These studies suggest that NO levels may prove useful as a means of early detection of subclinical inflammation in individuals who are working in environments that may increase their risk of asthma.
Exhaled NO in COPD
To date, assessments of exhaled NO in patients with COPD have provided seemingly conflicting results. Maziak and coworkers showed that patients with COPD, particularly those with unstable disease, had higher levels of exhaled NO than smokers with chronic bronchitis.292 These results were supported by Kanazawa et al. who found that exhaled NO levels were higher in patients with COPD than in healthy controls (12.1 ± 1.9 ppb vs.
5.2 ± 1.4 ppb).293 Others have reported similar results.294,295 Ansarin and coworkers reported that NO levels were higher in patients with COPD than in controls, but were lower than in patients with asthma.122 NO levels correlated with lung function tests in this study.
In a study by Clini and colleagues, however, patients with severe but stable COPD have been shown to have abnormally low levels of exhaled NO.296 Other studies by the same group have shown that NO levels were significantly lower in COPD patients with cor pulmonale than in those patients who did not have this complication (5.7 ± 1.9 ppb vs.
8.9 ± 4.7 ppb)297 and pulmonary rehabilitation in patients with mild to moderate COPD was associated with an increase in exhaled NO.159 Furthermore, Rutgers et al.298 and Delen et al.120 both reported no differences in NO levels between patients with stable COPD and healthy controls. AERO003-Proof 04 7/4/05 5:37 pm Page 44 A possible explanation for these conflicting results may be differing inflammation pathologies in patients with COPD. This condition is recognized as an inflammatory disorder associated with sputum neutrophilia and, in some cases, eosinophilia. In patients with fixed airflow obstruction, Fabbri et al. have shown that subjects with a history of asthma have significantly more eosinophils in blood, sputum, BAL and airway mucosa than patients with a history of COPD. Exhaled NO was also significantly higher in the patients with a history of asthma, 37.5 ppb compared with 11.1 ppb (flow rate not stated) in the patient group with a history of COPD (P < 0.01; Figure 38).186 Figure 38. Patients with a history of asthma had higherexhaled NO levels than those with a history of COPD Another study by Clini et al. showed ( P < 0.01). Horizontal solid bars indicate the median valuefor each group. Asterisks indicate a significant difference that exhaled NO and peak work rate between patients with a history of asthma and patients with increased in patients with COPD of a history of COPD186 differing severity after a pulmonary Machado et al. have shown exhaled NO to be lower in patients with β1-antitrypsin deficiency than in COPD patients without this deficiency.299 Some patients with COPD respond to corticosteroids whereas others do not. One study suggests that patients who respond to these anti-inflammatory agents tend to be those with higher counts of eosinophils.300 In another study, patients with COPD who experienced partial reversibility of airflow limitation after salbutamol treatment had higher levels of sputum eosinophils than those who showed no response to this bronchodilator.97 Furthermore, those who showed a partial response had higher levels of exhaled NO compared with healthy controls and those who showed no response (Figure 39). NO levels have been Figure 39. Exhaled NO levels in COPD patients whorespond to a bronchodilator (COPD–REV), those who fail shown to decrease in response to to respond (COPD–nonREV), and in healthy controls.
Horizontal bars represent median values. ** P < 0.01 corticosteroids in COPD patients.121 compared with control97 AERO003-Proof 04 7/4/05 5:37 pm Page 45 It is likely that COPD may have different pathogeneses. One pathogenesis may be associated with more extensive eosinophilic inflammation and increased NO levels.
Interestingly, one study has shown that various factors, including exhaled NO, airflow limitation and sputum eosinophils and neutrophils are separate and largely independent components of COPD pathophysiology.301 It is possible that exhaled NO may have the potential to distinguish COPD patients who will respond to anti-inflammatory treatment. VII. Exhaled NO in Smokers
Smokers have an increased risk of experiencing respiratory infections and pulmonary–vascular complications, in addition to chronic respiratory disorders. A lack of endogenous NO may play a role in the increased risk of some of these disorders.
Substantial evidence is available to show that exhaled NO levels are reduced in smokers.
For example, Kharitonov and coworkers showed in an early study that exhaled NO levels were over 50% lower in smokers compared with nonsmokers.302 Others have reported similar results.144,145,303 Nasal NO levels are also reduced in smokers.303 Interestingly, exhaled NO levels are higher in smokers with asthma than in healthy smokers, suggesting that exhaled NO may still be a useful marker of airway inflammation in smokers.146 Passive smoking may also reduce exhaled NO levels. Maniscalco et al. reported that exhaled NO levels in healthy individuals fell from 16.7 ± 1.4 ppb to 13.9 ± 1.33 after short- term exposure to environmental cigarette smoke.149 However, the decrease was transient, recovering within 30 minutes. Yates and colleagues also found a temporary decrease (by 23.6%) in NO levels after exposure to environmental cigarette smoke, which was significant compared with a decrease following sham exposure.148 Notably, active cigarette smoking was associated with a decrease in exhaled NO that remained low. Both these studies involved adults. However, one study involving children suggests that exhaled NO levels were not reduced in healthy individuals exposed to tobacco smoke.150 This study also assessed exhaled NO levels in children with asthma and found that, in this case, passive smoking was associated with a decrease.
The cause of the decrease in NO levels associated with smoking requires further investigation. However, in vitro research suggests that cigarette smoke decreases the activity of iNOS in lung epithelial cells304 and eNOS in pulmonary artery endothelial cells.305 It has been suggested that cigarette smoke must in part reduce NO levels produced in the oropharyngeal tract. However, it has been shown that iNOS expression is not reduced in oropharyngeal biopsies, nor is there evidence of reduced nonenzymatic formation of NO in this region.145 In a study by Högman et al., diffusion modeling showed that NO flux from the airways was reduced in smokers, but alveolar NO was increased.144 AERO003-Proof 04 7/4/05 5:37 pm Page 46 Whatever the mechanism affecting NO levels in smokers is, it does appear to be reversible. In the Högman study, some patients stopped smoking for 4 weeks with the result that airway NO flux increased to levels not dissimilar from nonsmokers.144 Another study showed that exhaled NO levels increased after just 1 week of not Mean exhaled NO (ppb) smoking and had increased again by 8 weeks (Figure 40).303 Baseline 1 week 8 weeks Healthy individuals who failed to stop smoking did not show an increase. These reports Figure. 40. Mean exhaled NO levels (10 mL/s) in smokers suggest that measurement of exhaled NO following cessation of smoking and in healthy controls303 levels may have a role in smoking cessation programmes. An increase in NO levels may be a simple method of demonstrating improvement to an individual and thus encourage continued abstinence. In addition, NO levels may highlight lack of compliance with a cessation programme.
VIII. Exhaled NO in Other Diseases
Assessment of Chronic Cough
Accurate diagnosis of patients complaining of chronic cough is essential for the underlying disease to be treated correctly. In more than 90% of cases, symptoms are a result of smoking, postnasal drip, gastroesophageal reflux, asthma or COPD.
Chatkin et al.93 investigated the value of exhaled NO in the assessment of chronic cough. Exhaled NO was measured in adults with chronic cough, known asthmatics and healthy controls. Patients rue positive rate (%)T with chronic cough and asthma had significantly higher exhaled NO values than non-asthmatics with chronic cough or healthy controls (Figure 41). Using 30 ppb (at a flow of 45 mL/s) as the cut-off point for Figure 41. A cut-off point of 30 ppb at an exhalation flow of45 mL/s provides a good specificity and sensitivity for the exhaled NO gave a sensitivity and detection of asthma93 AERO003-Proof 04 7/4/05 5:37 pm Page 47 specificity of 75% and 87%, respectively, for a diagnosis of asthma. Nogami et al. also described a significant negative correlation between exhaled NO levels and bronchial hyper- responsiveness in patients with chronic cough.125 These findings are supported by other studies,77,306 one of which involved children aged 2–7 years.77 In this study, children with mild intermittent asthma had exhaled NO levels of 5.6 ± 0.4 ppb (flow rate not specified) compared with 3.2 ± 0.3 ppb in those with chronic cough and 2.2 ± 0.2 in healthy individuals. As in asthma, inhaled corticosteroids have been shown to reduce exhaled NO levels in patients with chronic cough. In one study involving 88 patients, the reduction in NO levels was accompanied by a modest improvement in the severity of cough.127 The relationship between coughing frequency and exhaled NO levels has also been investigated in children with asthma. Li et al. showed that the cough frequency in children with stable asthma was increased compared with normal controls.126 The cough frequency was also found to have a significant positive correlation with exhaled NO levels (r = 0.781, P < 0.001; Figure 42) but not with FEV or sputum eosinophil count (r = –0.270, P = 0.157; r = 0.173, P = 0.508, In the clinical setting, these findings may make more invasive tests for asthma, Number of cough episodes such as bronchial challenge, unnecessary.
20 40 60 80 100 120 140 160 180 200 High values of NO are not specific for asthma, but make the diagnosis more likely and may help to indicate which patients Figure 42. There is a positive correlation between coughfrequency and exhaled NO levels in children with stable should be studied further.
asthma ( P < 0.001)126 Assessment of Cystic Fibrosis
Exhaled NO, elevated in most inflammatory airway diseases, is decreased in cystic fibrosis (CF), suggesting either decreased production or accelerated metabolism.72,307 Morrissey et al.
have shown that, despite confirmation of subnormal iNOS in the CF airway epithelium, the alternative isoforms nNOS and eNOS were present, and inflammatory cells in the CF airways expressed abundant iNOS. Increased immunohistochemical staining for nitrotyrosine was demonstrated in the lung tissue from patients with CF compared with controls.308 Using a 50 mL/s flow rate, exhaled NO in CF patients in this study was found to be 56% of that in AERO003-Proof 04 7/4/05 5:37 pm Page 48 normal volunteers. In a genetic study, Texereau and coworkers showed that patients with CF who had a NOS1 genotype associated with high NO production had a slower decline in lung function over 5 years.309 This suggests that the low NO levels seen in CF may be pathophysiologically related to poor lung function.
Exhaled NO levels have also been shown to be significantly lower in infants with CF compared with controls.310 However, another study in young children found no difference in NO levels compared with controls.311 In this study, iNOS expression decreased as airway inflammation increased; possibly indicating that low NO levels may be associated with worsening disease.
Grasemann and colleagues have shown that changes in exhaled NO levels parallel changes in pulmonary function in most patients treated with recombinant human DNase I, suggesting that NO levels may be a useful marker of efficacy.133 Allergic bronchopulmonary aspergillosis (ABPA) is a hypersensitivity reaction to Aspergillus fumigates (Af). Patients with CF may be prone to colonization with Af because of their highly tenacious sputum. Untreated ABPA may lead to lung damage, including proximal bronchiectasis, and segmental, lobar, or whole lung collapse. Furthermore, diagnosis of ABPA is difficult as the signs and symptoms are similar to those of CF itself. Lim et al. have shown that exhaled NO levels were lower in CF patients on glucocorticoids with a high risk of developing ABPA than in those with a lower risk.132 Therefore, exhaled NO levels may be a useful predictor of CF patients who are at risk of developing ABPA.
NO Levels and Transplantation
Bronchiolitis obliterans, a major mid- and long-term complication of lung transplants is a chronic inflammatory disorder affecting predominantly the terminal and respiratory bronchioles. It causes an obstructive syndrome that subsequently leads to graft failure.
Diagnosis of obliterative bronchiolitis (OB) and BOS depends on changes in pulmonary function after lung transplantation (a gradual decrease in FEV or FEF in mid-expiratory phase), with or without pathological proof of OB.312 Using these criteria, BOS can be diagnosed only when a loss of ≥ 20% of the initial best postoperative FEV has occurred.
Consequently much research is now focused on the establishment of early markers for chronic rejection after lung transplantation. Exhaled NO is also being investigated as a potential marker.
Exhaled NO levels were shown in a rat model of acute lung transplant rejection to be elevated in fulminant acute rejection.116 Exhaled NO levels correlated with the degree of acute lung allograft rejection.
AERO003-Proof 04 7/4/05 5:37 pm Page 49 An increase in exhaled NO in lung transplant recipients with chronic rejection has already been demonstrated.313,314 A preliminary study involving lung and cardiac transplant patients showed that single-breath exhaled NO levels were strikingly different in patients suffering from BOS.315 In these patients, the decrease from peak-to-end expiratory NO concentration was slower. At mid-expiration, NO levels were approximately three times higher in the BOS group. The increase in mid-expiratory NO levels did not appear to be related to the syndrome itself. High exhaled NO levels have been shown recently to be a strong diagnostic marker for BOS. Thirty-two patients were followed for up to 2 years after receiving a lung transplant and 13 developed BOS.119 All but one of the patients who developed BOS had two consecutive NO measurements of ≥ 15 ppb in the months preceeding the diagnosis. In comparison, onlythree of the 19 patients who did not develop BOS had such NO measurements – resulting in a diagnostic accuracy of 88%.
Gabbay and colleagues showed that exhaled NO levels, although normal, reflected the degree of airway inflammation in 20 stable lung transplant recipients (mean age 49 ± 3 years).316 Using regression analysis they showed that the percentage of bronchiolar lavage neutrophils (r2 = 0.82; P < 0.0001) and iNOS expression in the bronchial epithelium (r2 = 0.75; P < 0.0001), but not in the lamina propria (r2 = 0.16; P = 0.08), were positively predictive of exhaled NO levels. The same group went on to show that in BOS, exhaled NO levels were increased in association with even greater airway neutrophilia and enhanced expression of iNOS in the bronchial epithelium.117 Verleden and colleagues demonstrated that a switch from cyclosporine to tacrolimus therapy stabilized FEV patients with chronic rejection levels Months before and after switch accompanied by a decrease in exhaled NO (Figure 43).317 These results suggest that exhaled NO level measurements can be valuable in guiding the treatment of chronic rejection after lung transplantation. The same group also showed that there was no significant difference in exhaled NO levels between patients with chronic rejection who Months before and after switch underwent single lung transplantation and those who underwent sequential single Figure 43. After switching treatment from cyclosporine totacrolimus there was (a) a significant decline in FEV , with lung transplant and heart/lung stabilization after Time 0 (** P = 0.0047); (b) a gradualdecline in exhaled NO levels after Time 0 in the whole AERO003-Proof 04 7/4/05 5:37 pm Page 50 Nasal NO Measurements
Just as orally exhaled NO has been studied as a marker of airway inflammation, nasal NO has been studied as a marker of nasal inflammation.3 In the nose, NO is thought to play a role in host defence through its own antimicrobial and antiviral activity and by up-regulating ciliary motility.318,319 Most of the NO in nasal air is derived from epithelial cells in the paranasal sinuses, with concentrations in the sinuses being close to the maximum allowed atmospheric levels.53,54 It is thought that the high levels of NO in the sinuses play a role in maintaining sinus sterility.
Like exhaled NO, guidelines are available for nasal NO measurements.52 Generally, nasal NO levels are much higher than exhaled NO levels, with values over 100 and up to 2000 ppb having been reported in healthy individuals (Table 6).71,100,107,108,320–327 Table 6. Nasal NO levels in healthy individuals.
Author, year
Nasal NO level (ppb)
Tornberg et al., 2003100 Cervin et al., 2002322 Dotsch et al., 199671 Lindberg et al., 1997323 Grasemann et al., 1999324 Horváth et al., 2003108 Karadag et al., 1999320 Loukides et al., 1998325 Narang et al., 2002107 Noone et al., 2004326 Wodehouse et al., 2003327 Cervin et al. have shown nasal NO measurements to decrease within 10 minutes after nasal challenge with histamine and after challenge with the vasoconstrictor oxymethazoline in healthy non-allergic subjects.322 Both plasma exudation and nasal blockade increased within 10 minutes of the challenge, indicating that the different signs of airway inflammation are not directly linked and may reflect different aspects of nasal mucosal inflammation.
Tornberg et al. also showed that nasal NO levels are significantly higher in patients during anaesthesia (315 ± 34 ppb) than while awake (177 ± 17 ppb; P = 0.01).100 A study carried out on premature infants (median gestational age of 27 weeks), showed that nasal NO could be detected directly from the nasal space using a chemiluminescence analyser.328 Lower airway NO was also sampled from a catheter positioned so that its tip lay at the lower end of the endotracheal tube. Nasal NO levels were higher than lower airway AERO003-Proof 04 7/4/05 5:37 pm Page 51 NO levels, even on the first day after birth, showing that care must be taken to avoid contamination with nasal NO when assessing lower airways accurately. Nasal and lower airway NO levels did not correlate significantly with gestational age, but lower airway NO levels correlated with postnatal age (r = 0.86, P = 0.014).
Although attempts to standardize nasal NO measurements have been made, detecting alterations in nasal output is limited by the high background NO levels in the upper airways originating from several sources in the nose and sinuses. In particular, the paranasal sinuses seem to be major sources of NO and local concentrations can reach well over 20 ppm.53 Nasal humming has been shown to speed up the exchange of air between sinuses and the nasal cavity thereby dramatically increasing nasal NO output.329,330 Maniscalco and colleagues showed that during repeated humming manoeuvres an initial NO peak is observed followed by a progressive decline (Figure 44).330 This is probably because most sinus NO has been washed out. The same group also showed in a different study that nasal NO levels measured immediately after repeated humming manoeuvres are consistently lower and more reproducible than nasal NO levels measured after a period of silence or free speaking, probably due to the sinus NO contribution being temporarily decreased and thereby removing the variability in NO measurements conferred Humming exhalation by nasal NO.331 Mean nasal NO output Silent exhalation (95% CI) after a period of silence/free speaking was 231 nL/min (range 178–284 nL/min) in healthy volunteers, 434 nL/min (range 347–522 nL/min) in patients with allergic rhinitis (P < 0.001), and 262 nL/min NO output (nL/min) (163–361 nL/min) in patients with allergic nasal polyposis. The authors concluded by suggesting that repeated humming manoeuvres could be useful to better distinguish nasal mucosal NO output from Figure 44. During nasal humming an initial NO peak is that of the paranasal sinuses.
observed followed by a progressive decline330 Several studies have indicated that nasal NO levels are increased in patients with allergic rhinitis.332–334 For example, Kharitonov and coworkers reported nasal NO levels to be 1527 ± 87 ppb in untreated patients with allergic rhinitis, whereas levels of 996 ± 39 ppb were found in healthy controls.333 In addition, nasal NO levels were lower in those patients who received nasal corticosteroid treatment. Similar results have been reported by other groups.332,335 AERO003-Proof 04 7/4/05 5:37 pm Page 52 However, other groups have shown no significant difference in nasal NO levels between patients with rhinitis or similar nasal symptoms and healthy individuals.269,336 In addition, some authors have found no effect of treatment on nasal NO levels in patients with allergic rhinitis.337 Whether nasal NO levels are increased in rhinitis is still open to debate. Given that most nasal NO originates in the sinuses, a more conclusive study regarding rhinitis would involve measurement of nasal NO that excluded sinus NO. Currently no technique exists for this measurement, although measurements by nasal humming may be an option. It has been shown that nasal epithelial cells from patients with rhinitis express higher levels of iNOS than cells from healthy individuals, suggesting the presence of higher NO levels in affected Rhinovirus infections are associated with unpleasant symptoms and exacerbate asthma and COPD. NO has potent antiviral properties and thus may have a role in host defense. If this is the case, one would expect nasal NO levels to be increased in individuals infected with Sanders and coworkers investigated nasal NO levels in six volunteers who were infected with rhinovirus-16.113 NO levels increased significantly and were associated with increased expression of iNOS mRNA in nasal scrapings. Furthermore, the increase in nasal NO correlated inversely with symptom scores, possibly indicating an antiviral role. The group had previously shown that rhinovirus increased iNOS mRNA expression in vitro and in Primary Ciliary Dyskinesia
An area where nasal NO measurements may prove particularly useful is in the diagnosis of PCD. Karadag and colleagues reported that nasal NO levels in patients with PCD were much lower than in healthy individuals (97 ppb vs. 664 ppb).320 Similarly, Baktai and coworkers found that the concentration of nasal NO in patients with PCD characterized by lack of both outer and inner dynein arms was 36.2 ppb, whereas healthy controls had levels of 1047 ppb.105 Interestingly, patients who had PCD with a lack of inner dynein arms only had much higher NO levels (869 ppb) than the other affected patients. Others have also confirmed that PCD is associated with extremely low levels of nasal NO106,340 and these low levels seem highly predictive of the disease. Narang and colleagues studied exhaled and nasal NO in 102 children attending a respiratory medicine clinic and compared their results with those of 53 healthy controls.107 The children with respiratory disease included patients with PCD, asthma, CF and non-CF bronchiectasis. Nasal air was sampled from one nostril during breath-hold, using a sampling AERO003-Proof 04 7/4/05 5:37 pm Page 53 rate of 250 mL/min. Nasal NO levels were significantly lower in PCD patients than in all the other groups. Median values were 60.3, 533.6, 491.3, and 716.0 ppb in children with PCD, bronchiectasis, CF, and controls, respectively (P < 0.05). Only one patient with PCD had a nasal NO level greater than 250 ppb, and 80% had nasal NO levels of less than 100 ppb. There was some overlap with patients with CF and bronchiectasis, but the authors calculated that nasal NO levels of less than 250 ppb had a positive predictive value of 83% and a negative predictive value of 97% of identifying patients with PCD. Sensitivity and specificity for PCD at various cut-off points for nasal NO are shown in Figure 45.
1 – Specificity In another study NO levels < 105 ppb had a positive predictive value of 89% and a Figure 45. Receiver-operator characteristic curve showing negative predictive value of 100%.109 the value of various nasal NO concentrations in thediagnosis of PCS107 Horváth and colleagues found similar results in their study.108 They measured exhaled and nasal NO levels in bronchiectatic patients with PCD (n = 14), non-PCD bronchiectatic patients with CF (n = 20) and without CF (n = 31), and healthy volunteers (n = 37). Nasal NO levels were significantly lower in PCD patients than in any other group (PCD: 54.5 [5.0–269] ppb; non-PCD bronchiectasis without CF: 680 [310–1000] ppb; non-PCD bronchiectasis with CF: 343 [30–997] ppb; control: 663 [322–1343] ppb). Exhaled NO levels followed the same pattern, but levels were not lower than in bronchiectatic patients with CF.
The reason for these low NO levels may be due to a deficiency in iNOS in nasal epithelium.341 Furthermore, this deficiency may be linked to the genetic cause of the PCD, as nasal NO is low in healthy carriers of mutations associated with the disease, but not as low as values seen in affected patients.342 PCD can be difficult to diagnose and thus nasal NO measurements are useful in diagnosing the condition,343,344 particularly as the test is simple to As with exhaled NO measurements, nasal NO levels are lower than normal in individuals with CF, although not to the same extent as seen in PCD.72,345 Nevertheless, nasal NO measurements may prove helpful in the differential diagnosis of this condition. AERO003-Proof 04 7/4/05 5:37 pm Page 54 Flow-Independent Parameters: Based on NO
From a pathophysiological viewpoint, it would be useful to know where inflammation associated with exhaled NO is located (i.e. the distribution in the bronchi and alveoli) and how far the inflammation extends into the peripheral airways. It would also be interesting to see if the diffusion capacity is related to different disease states. NO diffusion models have the potential to provide this information.
It can be assumed that the concentration of NO in the alveolar compartment is constant relative to the variable concentration in the airways, due to the changing flow rate. During an exhalation, as air from the alveolar compartment moves through the bronchial compartment, NO from the bronchial wall will diffuse into the airway lumen, thereby increasing the NO concentration. If the exhalation is slow, there will be time for NO to diffuse into the airway, thus increasing the concentration to a greater extent than if the exhalation is fast. This basic concept can therefore easily account for the flow dependence of NO measurements.346 NO Diffusion Models
It is not feasible to draw up a model that accounts for the whole complexity of the lungs.
However, one can simplify by using a model that assumes that all alveoli can be united in one compartment and all bronchi in a second compartment, which forms a sort of pipe connected to the alveolar compartment. This partitioning of the alveolar and airway components is referred to the two-compartment model.347 This approach takes advantage of the fact that the relationship between NO output and expiratory flow appears to be linear above a threshold of 50 mL/s.347,348 Similar models followed the two- compartment model, as described by Pietropaoli et al.,349 Silkoff et al.350 and Högman et al.351 In addition, new breathing techniques that use the two- compartment model have been developed to characterize NO exchange dynamics (Figure 46).352,353 Alveolar region Airway region (Compartment 1) (Compartment 2) The most recent model published is by Shin and George who have incorporated axial diffusion into a one-dimensional Figure 46. NO diffusion model. C = steady-state alveolar = max influx of NO from the airways, trumpet model of NO gas exchange in the = exhaled NO concentration, D capacity of NO in the airways, C = concentration of NO lungs.354 The trumpet model predicts a in gas phase within the airway region353 AERO003-Proof 04 7/4/05 5:37 pm Page 55 significant back-diffusion of NO from the airways into the alveolar region, resulting in a significant loss of NO that would therefore not appear in the exhaled breath. Furthermore, this back-diffusion of NO means that there is far less partitioning between alveolar and airway NO than assumed in the two-compartment model.355 The result is a potential underestimation of both the maximum airway flux of NO and the airway diffusing capacity for NO. This effect will, however, only be significant if there is substantial production of NO in the very small airways.
All these models assume that exhaled NO levels increase as exhalation flow decreases, because of prolonged contact of the expirate with bronchial epithelium. This raises the intriguing possibility of whether variations in inspiration rate could contaminate alveolar NO with bronchial-derived NO, and thus affect exhaled NO measurements. This has recently been investigated by Zacharasiewicz et al., but they found no effect of different inhalation rates.356 Thus, we can be assured that current techniques for measuring exhaled NO do not need to take inhalation rates into consideration.
Clinical Use
Diffusion models are now beginning to provide valuable information into a wide range of inflammatory diseases, as outlined below. Further insights are expected as this relatively new area continues to develop.
Studies indicate that diffusing capacity of the airways is increased in patients with asthma.
Silkoff et al. found the diffusion capacity in patients with asthma was four times that of healthy individuals.350 Notably, inhaled corticosteroids had no effect on diffusion capacity.
These results were confirmed by Shin and coworkers using a different technique.357 This increase in diffusion capacity may reflect upregulation of non-adrenergic, non- cholinergic, NO-producing nerves in airways in compensation for decreased sensitivity of airway smooth muscle to the relaxed effects of endogenous NO.350 Interestingly, diffusion capacity has been shown to correlate inversely with FEV and FVC, suggesting that this parameter may reflect physiological changes in the airways that do not respond to inhaled Bronchial NO flux also appears to be relatively high in patients with asthma. In the study by Silkoff and coworkers, NO flux was 6512 pL/s in patients with asthma who were not receiving corticosteroids, compared with 1020 pL/s in healthy controls.350 Corticosteroid treatment was associated with a reduction in NO flux (2416 pL/s) and the parameter was correlated to lung function. Again, Shin et al. were able to confirm the general findings of increased and corticosteroid-responsive bronchial flux, but found no correlation with lung function tests.357 Data from another group also suggest no correlation between bronchial flux AERO003-Proof 04 7/4/05 5:37 pm Page 56 and lung function.358 Although it is clear that corticosteroids have an impact on bronchial flux, how this reduction in flux affects symptoms and long-term outcomes requires further Alveolar NO does not appear to be consistently increased in asthma. Lehtimaki et al.
found that alveolar NO levels were similar to healthy individuals and patients with asthma, but were significantly increased in patients with asthma symptoms.359 The same group report that alveolar NO levels are increased in asthma patients with nocturnal symptoms, but not in those who do not have nocturnal asthma.360 Others have also found that alveolar NO is only increased in asthma patients with recent symptoms.361 However, Glelb and colleagues demonstrated a significantly higher alveolar NO concentration in patients with asthma compared with controls (7.0 ppb vs. 3.2 ppb, P = 0.01). Alveolar NO has been shown to correlate to airway remodeling as assessed by TGF-β levels in bronchoalveolarlavage fluid and inflammatory markers.362 Furthermore, alveolar NO levels appear to respond to oral corticosteroid treatment in patients with asthma refractory to inhaled Patients with CF have an increased diffusing capacity of NO in the airways, decreased mean tissue concentration of NO in the airways, and decreased steady-state alveolar concentration compared to healthy age-matched children.363 Allergic Alveolitis
Bronchial NO flux is increased in asthma in comparison with alveolitis and healthy controls.
Alveolar NO concentration is higher in alveolitis than in asthma and healthy controls.115 Girgis et al. confirmed that bronchial flux of NO was increased in scleroderma (SSc), regardless of whether interstitial lung disease (ILD) and pulmonary hypertension were present.364 Alveolar NO was also increased in SSc, regardless of whether ILD or pulmonary hypertension was present. Patients with liver cirrhosis have been shown to have an increased alveolar NO concentration (8.3 ± 0.9 ppb) compared with healthy subjects (4.7 ± 0.3 ppb).365,366 In patients who have cirrhosis associated with Sjogren syndrome, bronchial flux is also increased.366 Sjorgren syndrome is an autoimmune disorder that is often associated with other autoimmune conditions. Bronchial flux, but not alveolar NO, is increased in this condition.366 AERO003-Proof 04 7/4/05 5:37 pm Page 57 Using a model based on the classic Fick's first law of diffusion to partition NO in the lungs, the alveolar levels of NO in COPD patients were found to be increased (4 ± 2 ppb; P < 0.001) compared with control patients.367 Clinical Relevance of NO Measurements
As a highly sensitive and rapidly reactive marker of response to steroid treatment, measurement of exhaled NO levels offers a valuable means of monitoring the efficacy of asthma treatment.203,214 Exhaled NO levels have been shown to fall rapidly after the start of steroid treatment in both acute and chronic asthma,75,198,226–229,231,239,240,242 and repeated measurements during the treatment of asthma offers an easy but reliable method of tracking progress, particularly as evidence is growing that NO levels can predict loss of asthma Failure of exhaled NO levels to respond rapidly to adequate corticosteroid treatment in asthma suggests either steroid unresponsiveness274 or poor compliance with treatment.256,258,273 Exhaled NO monitoring would aid early identification of these scenarios.
In the study by Green et al., steering the anti-inflammatory dose towards normalizing the patient's inflammation status (sputum eosinophils) resulted in a healthier patient.277 Treatment based on the inflammatory marker resulted in fewer severe exacerbations and fewer emergency room visits, forming the proof of concept for the utility of inflammatory markers in routine clinical practice.
Exhaled NO is a non-specific indicator of airway inflammation. Levels are also raised in other conditions such as upper respiratory tract infection, tuberculosis, bronchiectasis and COPD. However, exhaled NO levels can help to distinguish asthma from chronic cough, The recent comparison of exhaled NO measurements with current conventional clinical tests recommended by international guidelines in the diagnosis of asthma concluded that exhaled NO measurements were superior to conventional approaches.170 Furthermore, measuring exhaled NO levels was advantageous as the test was quick and easy to perform.
Similar to exhaled NO measurements, nasal NO assessments may be useful in monitoring rhinitis and its treatment. In addition, nasal NO measurements offer a simple method of screening for PCD and may also help to distinguish it from CF.
AERO003-Proof 04 7/4/05 5:37 pm Page 58 XII. Summary
• Exhaled NO is elevated in asthma and correlates with generally accepted clinical markers of airway inflammation,3,168,181,184,197–199 particularly the results of bronchial biopsies.178,179 • Levels of exhaled NO in patients with asthma appear to correlate with disease • Treatment of airway inflammation in asthma with systemic or inhaled corticosteroids reduces levels of NO in exhaled air.75,175,214,224,226–229,231,234,240,242 • Anti-inflammatory treatment with inhaled leukotriene antagonists lower exhaled NO • Persistently elevated levels of exhaled NO in asthmatics treated with inhaled corticosteroids suggest poor compliance with treatment.203,218,256,258,273,274 • Successive measurements of exhaled NO can be used to monitor the level of airway inflammation, predict loss of control and guide treatment with corticosteroids.4,188,197,231 • Increase in exhaled NO in patients with asthma precedes clinical exacerbation.175,188,275 • Exhaled NO may be raised in individuals before symptoms occur.34,188,278,288,289 • Patients with COPD who have high levels of exhaled NO may be more likely to respond to corticosteroid treatment.97 • Measurement of exhaled NO is an easy, non-invasive procedure that can be performed in adults and children.52,63 • Published guidelines allow standardization of the measurement of exhaled NO.51,52 • A number of studies have shown normal exhaled NO values (at a flow rate of 50 mL/s) to be 5–30 ppb (see Table 2) • The determination of NO provides diagnostic value and the ability to differentiate between healthy subjects and patients with asthma.29,180,264,267 NO measurements have been shown to be superior to current conventional clinical tests recommended by international guidelines in the diagnosis of asthma.170 AERO003-Proof 04 7/4/05 5:37 pm Page 59 • Measurement of exhaled NO may provide a useful method of differentiating asthma from XIII. Further Reading
The following papers provide excellent concise reviews of many of the topics covered in this • Smith and Taylor Curr Opin Allergy Clin Immunol 2005; 5: 49–56368 • Dinakar Curr Allergy Asthma Rep 2004; 4: 454–9369 • Malmberg J Asthma 2004; 41: 511–20370 • Kharitonov Swiss Med Wkly 2004; 134: 175–92371 • Bates and Silkoff J Allergy Clin Immunol 2003; 111: 256–62372 AERO003-Proof 04 7/4/05 5:37 pm Page 60 To keep up to date with the most recent advances in exhaled NO, log into www.aerocrine.com/references. The Exhaled Nitric Oxide Publication Reports cover all publications found within the PubMed database relating to ‘Exhaled/Expired Nitric Oxide'.
To date there are >1000 publications on this topic.
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232330 Scient Oms 03 05-04-21 08.00 Sida 2 NIOX® is CE-marked according to Medical Device Directive MDD 93/42/EEC and approved for clinical use in EEC countries. NIOX® is 510(k) cleared with FDA for clinical use in the US. tified according to ISO 14001 US patent 5,447,165, US patent 5,922,610, US patent 6,038,913, US patent 6,063,027, US patent 6,099,480, US patent 6,149,606, US patent 6,183,416, US patent 6,511,425, US patent 6,626,844, US patent 6,723,056, US patent 6,761,185 and patents pending.
NIOX MINO™ is a trademark and NIOX® is a registered trademark of Aerocrine AB.
Based on the company's intellectual property, Aerocrine develops and commercializes products for the monitoring of nitric oxide (NO) as a marker of inflammation, to improve the management and care of patients with inflammatory disease in the airways.
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