Kartagener syndrome

Study objectives: To investigate whether nitric oxide (NO) metabolites would be reduced in children affected by primary ciliary dyskinesia (PCD).

Design: Single-center observational study.

Patients: Fifteen children with PCD (seven boys; mean [[+ or -] SEM] age, 10.3 [+ or -] 0.7 years; mean [FEV.sub.1], 73 [+ or -] 2.1% predicted) were recruited along with 14 healthy age-matched subjects (seven boys; mean age, 11.5 [+ or -] 0.4 years; mean [FEV.sub.1], 103 [+ or -] 5% predicted).

Interventions: We assessed the levels of nitrite (N[O.sub.2.sup.-]), N[O.sub.2.sup.-]/N[O.sub.3.sup.-] (N[O.sub.2.sup.-]/N[O.sub.3.sup.-]), and S-nitrosothiol in exhaled breath condensate, exhaled NO, and nasal NO from children with PCD compared to those in healthy children.

Measurements and results: The mean exhaled and nasal NO levels were markedly decreased in children with PCD compared to those without PCD (3.2 [+ or -] 0.2 vs 8.5 [+ or -] 0.9 parts per billion [ppb], respectively [p < 0.0001]; 59.6 [+ or -] 12.2 vs 505.5 [+ or -] 66.8 ppb, respectively [p < 0.001]). Despite the lower levels of exhaled NO in children with PCD, no differences were found in the mean levels of N[O.sub.2.sup.-] (2.9 [+ or -] 0.4 vs 3.5 [+ or -] 0.3 [micro]M, respectively), N[O.sub.2.sup.-]/N[O.sub.3.sup.-] (35.2 [+ or -] 5.0 vs 34.3 [+ or -] 4.5 [micro]M, respectively), or S-nitrosothiol (1.0 [+ or -] 0.2 vs 0.6 [+ or -] 0.1 [micro]M, respectively) between children with PCD and healthy subjects.

Conclusion. These findings suggest that NO synthase activity may not be decreased as much as might be expected on the basis of low exhaled and nasal NO levels.

Key words: breath condensate; exhaled nitric oxide; nitrite; nitrite/nitrate; primary ciliary dyskinesia: S-nitrosothiols

Abbreviations: CBF = ciliary beat frequency; CF = cystic fibrosis; iNOS = inducible nitric oxide Synthase; NO = nitric oxide; NOS = nitric oxide synthase; N[O.sub.2.sup.-] = nitrite; N[O.sub.3.sup.-] = nitrate: ONO[O.sup.-] = peroxynitrite; PCD = primary ciliary dyskinesia; ppb = parts per billion

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Primary ciliary dyskinesia (PCD), including Kartagener syndrome, is a genetic disease that is characterized by defective motility of cilia, in which the levels of exhaled and nasal nitric oxide (NO) are very low compared to healthy subjects, (1-3) a Such low values of exhaled and nasal NO are not seen in patients with any other condition and are, therefore, of diagnostic value. (4,5)

The measurement of exhaled NO might be used as a screening procedure to detect PCD) among patients with recurrent chest infections or male infertility due to immotile spermatozoa, and the diagnosis of PCD would then be confirmed by the saccharine test, nasal NO level, ciliary beat frequency (CBF), and electron microscopy. (6) NO plays an important role in bactericidal activity in the lungs, sodium and chloride transport in the nasal epithelimn, and CBF, (7) so that a decrease in endogenous NO production might contribute to the characteristic recurrent chest infections in PCD patients, Low levels of exhaled and nasal NO in PCD patients are related to mucociliary dysfunction, (1,8) and treatment with NO donor L-arginine increases nasal NO levels and also improves mucociliary transport in PCD patients. (1,9) Considering the inflammatory nature of PCD, the markedly decreased exhaled and nasal NO levels are suprising, and the pathophysiologic basis of these observations are still not clear.

NO is a free radical that is rapidly oxidized, reduced, or complexed with other molecules depending on the microenvironment, leading to the formation of nitrite (N[O.sub.2.sup.-]), nitrate (N[O.sub.3.sup.-]), and powerful oxidant peroxynitrite (ONO[O.sup.-]). (10,11) ONO[O.sup.-] formation may be decreased in patients with PCD due to the NO deficit. It may further impair host defense in patients with PCD, as ONO[O.sup.-] may also nitrate inflammatory proteins such as the chemokines and, therefore, reduce oxidative damage. (12) In addition, ONO[O.SUP.-] interacts directly with glutathione to form the nitrososthiol S-nitrosoglutathione, which may protect against further effects of ONO[O.sup.-]. (13)

NO metabolites, such as N[O.sub.2.sup.-], N[O.sub.3.sup.-], and S-nitrosothiol, previously have been detected in exhaled breath condensate. (14) We speculated that the measurement of NO metabolites in exhaled breath condensate would provide further information about nitrogen-reactive species in the airways of patients with PCD. We hypothesized that, on the basis of the low levels of exhaled and nasal NO, exhaled NO metabolites would be low. The design of this study was to assess the levels of N[O.sub.2.sup.-] , N[O.sub.3.sup.-], and S-nitrosothiol in the exhaled breath condensate of children with PCD in comparison with healthy subjects.

MATERIALS AND METHODS

Patients

Patients were recruited from the pediatric PCD Clinic at the Royal Brompton Hospital. PCD was diagnosed by nasal brushing with an estimation of CBF and the determination of ultrastructural defects using electron microscopy. We studied 15 patients with PCD (seven boys; mean [[+ or -] SEM] age, 10.3 [+ or -] 0.7 years: age range, 7 to 14 years) who had a mean FE[V.sub.1] of 73 [+ or -] 2.1% of predicted. Eight patients were receiving therapy with inhaled corticosteroids. The age matched control group consisted of 14 healthy children (seven boys: mean age, 11.5 [+ or -] 0.4 years; mean FE[V.sub.1], 103 [+ or -] 5.0% of predicted) without a history, of chronic or recent acute, respiratory disease, who were healthy siblings of children who were attending the asthma clinic at Royal Brompton Hospital or were the children of staff members. The characteristics of the subjects are shown in Table 1. The protocol was approved by the Ethics Committee of the Royal Brompton and Harefield National Health Service Trust, and informed consent was obtained from all parents and children recruited into the study.

Study Design

Subject details were obtained, and then baseline spirometry. testing was performed, and exhaled NO and nasal NO levels were measured, followed by the collection of exhaled breath condensate.

Pulmonary Function

FVC percent predicted and FE[V.sub.1] percent predicted were measured with a dry spirometer (Vitalograph: Buckingham, UK), and the best value of three maneuvers was expressed as a percentage of the predicted value.

Exhaled NO Measurement

NO was measured by chemiluminescence analyzer (model LR2000; Logan Research Ltd: Rochester. UK) according to American Thoracic Society guidelines (15) and European Respiratory Society guidelines (16) on NO measurements, as described previously. (2,17)

Exhaled Breath Condensate

Exhaled breath condensate was collected by using a condenser, which allowed the noninvasive collection of nongaseous components of the expiratory air (EcoScreen; Jaeger; Wurzburg, Germany), as described previously. (14) Subjects breathed through a mouthpiece and a two-way nonrebreathing valve, which also served as a saliva trap. They were asked to breathe at a normal frequency and at tidal volume, wearing a nose clip, for a period of 8 min. The condensate, at least 700 [micro]L, was collected on ice at -20[degrees]C and was stored at -70[degrees]C immediately.

N[O.sub.2.sup.-], N[O.sub.2.sup.-] and N[O.sub.3.sup.-], and S-Nitrosothiol Measurements

The quantification of N[O.sub.2.sup.-] was assessed by a fluorometric assay based on the reaction of N[O.sub.2.sup.-] with 2,3-diaminonaphthalene to form the fluorescent product 1-(H)-naphthotriazole. (23) Briefly, the 100-[micro]L sample (exhaled breath condensate) was mixed with 10 [micro]L 0.05 mg/[micro]L 2,3 diaminonaphthalene reagent in 0.625 M HCl. The reaction was allowed to proceed at room temperature in the dark and was terminated with the addition of 10 [micro]L 1.4 M NaOH. The intensity of the fluorescent signal produced by the product was measured by a fluorometer (Biolite F1; Labtech International Ltd; Uckfield, UK) immediately. The incubation of samples with N[O.sub.3.sup.-] reductase allowed the N[O.sub.3.sup.-] present in the sample to be measured by this assay after being converted to N[O.sub.2.sup.-]. (14) S-nitrosothiols were measured following the release of N[O.sub.2.sup.-] from S-nitrosothiols by 2, mM [Hg.sub.2]Cl using the above mentioned procedure. (14) To calculate the level of S-nitrosothiols, N[O.sub.2.sup.-] levels were subtracted.

Statistical Analysis

Data were expressed as the mean [+ or -] SEM. A Mann-Whitney test was used to compare groups. The correlation between the fractional concentration of exhaled NO and N[O.sub.2.sup.-], N[O.sub.2.sup.-] /N[O.sub.3.sup.-], and S-nitrosothiol, as well ms NO metabolites and lung function (ie, [FEV.sub.1]) was determined by nonparametric Spearman correlation analysis. Significance was defined as a value of p < 0.05.

Lower and Upper Airway NO

The mean exhaled NO levels were significantly decreased in PCD patients compared to those in healthy subjects (3.2 [+ or -] 0.2 vs 8.4 [+ or -] 0.9 parts per billion [ppb], respectively; p < 0.0001) [Fig 1]. There was no significant difference between the steroid-naive and steroid-treated groups (3.4 -+ 0.3 vs 3.0 [+ or -] 0.3 ppb, respectively). There was no correlation between exhaled NO and [FEV.sub.1], N[O.sub.2.sup.-], N[O.sub.2.sup.-]/N[O.sub.3.sup.-] and S-nitrosothiol levels in exhaled breath condensate. Upper airway NO levels also were found to be markedly reduced in children with PCD compared to healthy control subjects (59.6 [+ or -] 12.2 vs 505.5 [+ or -] 66.8 ppb, respectively; p < 0.001).

[FIGURE 1 OMITTED]

N[O.sub.2.sup.-], N[O.sub.2.sup.-]/N[O.sub.3.sup.-], and S-Nitrosothiols in Breath Condensate

There was no significant difference in the levels of N[O.sub.2.sup.-] (2.9 [+ or -] 0.4 vs 3.5 [+ or -] 0.3 [micro]M, respectively), N[O.sub.2.sup.-]/N[O.sub.3.sup.-] (35.2 [+ or -] 5.0 vs 34.3 [+ or -] 4.5 [micro]M, respectively), and S-nitrosothiol (1.0 [+ or -] 0.2 vs 0.6 [+ or -] 0.1 [micro]M, respectively) in exhaled breath condensate between patients with PCD and healthy subjects. No differences were found in the levels of N[O.sub.2.sup.-], N[O.sub.2.sup-]/ N[O.sub.3.sup.-], and S-nitrosothiol (2.8 [+ or -] 0.7 vs 3.1 [+ or -] 0.5 [micro]M, respectively; 34.1 [+ or -] 6.6 vs 36.5 [+ or -] 8.1 [micro]M, respectively; and 1.0 [+ or -] 0.3 vs 1.0 [+ or -] 0.2 [micro]M, respectively) in exhaled breath condensate from steroid-naive and steroid-treated PCD patients and between patients who were or were not receiving long-term antibiotic treatment (3.2 [+ or -] 0.7 vs 2.6 [+ or -] 0.5 [micro]M, respectively; 34.2 [+ or -] 7.9 vs 36.3 [+ or -] 6.3 [micro]M, respectively; and 1.3 [+ or -] 0.3 vs 0.6 [+ or -] 0.2 [micro]M, respectively). There was no correlation among N[O.sub.2.sup.-], N[O.sub.2.sup.-]/ N[O.sub.3.sup.-], and S-nitrosothiol levels in exhaled breath condensate, and also no correlation was found among exhaled NO levels, [FEV.sub.1] and N[O.sub.2.sup.-], N[O.sub.2.sup.-]/ N[O.sub.3.sup.-], and S-nitrosothiol levels.

DISCUSSION

This study was designed to investigate whether the levels of NO metabolites, such as N[O.sub.2.sup.-], N[O.sub.2.sup.-]/ N[O.sub.3.sup.-], and S-nitrosothiol, in exhaled breath condensate were reduced in PCD patients, as might be expected from the lower levels of exhaled and nasal NO that have been described previously. (1,2) Surprisingly, no differences were found in the levels of exhaled N[O.sub.2.sup.-], N[O.sub.2.sup.-]/N[O.sub.3.sup.-], or exhaled S-nitrosothiol between patients with PCD and healthy subjects. There was a trend toward decreased exhaled N[O.sub.2.sup.-], levels in patients with PCD compared to healthy subjects. The levels of S-nitrosothiols were not decreased in PCD when compared to healthy subjects. Indeed, there was a tendency toward elevated levels.

The normal levels of NO metabolites cast doubt on the hypothesis that reduced exhaled and nasal NO levels are the results of reduced NO synthase (NOS) activity. Nevertheless, reduced NOS activity cannot be completely excluded. Normal NOS activity is comparable with a decreased level of exhaled NO if increased NO metabolism or reduced NO diffusion into the airway lumen occurs. Elevated levels of N[O.sub.2.sup.-] and N[O.sub.3.sup.-]. (18,19) and nitrotyrosine (20) have been found in the exhaled condensate and sputum (21) of patients with cystic fibrosis (CF) during clinical stability and during exacerbations. In children with CF and normal lung function, however, the N[O.sub.2.sup.-]/N[O.sub.3.sup.-] concentrations in BAL fluid are normal and concentrations of S-nitrosothiol are reduced. (22) In contrast, elevated levels of N[O.sub.2.sup.-] and S-nitrosothiol are found in the exhaled breath condensate of adult patients with more severe CF. (23) Therefore, it may be speculated that production/ metabolism changes may take place in PCD patients, so that the total NO production may seem to be similar to those of control subjects.

Myeloperoxidase, which is a heme enzyme of neutrophils that uses hydrogen peroxide to oxidize chloride to hypochlorous acid, is capable of catalyzing the nitration of tyrosine, providing an alternative to ONO[O.sup.-] in the formation of 3-nitrotyrosine. (24) At sites of neutrophilic inflammation, the presence of myeloperoxidase will lead to protein nitration because the cosubstrate tyrosine will be available to facilitate the reaction. (25) Patients with stable CF have significantly higher levels of nitrotyrosine in exhaled breath condensate than do healthy subjects. (20) This suggests that the nitration of proteins by myeloper-oxidase may be an additional source of nitrotyrosine in patients with CF who have very low NO production. In fact, the level of myeloperoxidase is elevated in the sputum of CF patients and correlates with concentrations of nitrotyrosine, (21) implying that the absence of an increase in exhaled NO does not exclude the possibility of NO participating in airway inflammation, including CF.

Free radicals released from neutrophils may increase NO metabolism by the conversion of NO to NO metabolites, such as N[O.sub.2.sup.-], N[O.sub.3.sup.-], and S-nitrosothiol, (26) and may lead to chronic, recurrent neutrophil inflammation, as seen in patients with CF and PCD. In fact, a positive correlation between the N[O.sub.2.sup.-] levels in exhaled breath condensate and the number of circulating plasma nentrophils in CF patients has been demonstrated. (18)

Some bacteria have been shown to produce NO from N[O.sub.2.sup.-]. (27) NO may play an important role in nonspecific host defense against bacterial, viral, and fungal infections. One of the general mechanisms of antimicrobial defenses involves the S-nitrosylation of NO by cysteine proteases. Therefore, reduced endogenous NO production, resulting in low exhaled and nasal NO levels, may contribute to recurrent chest infections in patients with PCD, CF, and Wegener granulomatosis. (28)

Patients with PCD are frequently or continuously treated with antibiotics, which can influence nasal and lower airway bacterial composition, which may influence NO production and NO metabolite levels in airway fluids. For example, it has been shown that benzoquinoid ansamycins were able to reduce N[O.sub.2.sup.-] accumulation, inducible NOS (iNOS) messenger RNA levels, and the cytokine-dependent activation of the iNOS promoter. (29) If this is the case, decreased exhaled NO levels would be associated with elevated levels of NO metabolites in airway fluids. However, our results do not support this hypothesis since no differences were found in the levels of exhaled NO metabolites between patients with PCD and healthy subjects. Furthermore, there were no differences either in the levels of exhaled NO or in the levels of NO metabolites in exhaled breath condensate between patients with PCD who were and were not receiving continuous antibiotic treatment.

Airway hygiene depends largely on mucociliary clearance and the movement of viscoelastic mucus along the airway by the beating of the ciliary append-ages of airway epithelial cells. (30) The failure to keep the airways sterile by mucociliary clearance, resulting in chronic damage to the airway wall and upregulation of mucus production, may be due to several of the following factors: (1) very low NO levels in PCD patients because of a deficiency of iNOS; (2) microbial toxin-induced dysfunction of the energy pathways required for ciliary beating (ie, secondary ciliary dyskinesia); and (3) abnormalities in the viscosity of mucus, including reduced salt content/osmolality, which results in it being unsuitable in quality for the cilia to move it. Therefore, methods of recognizing the prevalent mechanism behind the mucociliary clearance in PCD patients may be useful in disease management.

The effect of NO may be beneficial or deleterious, and both NOS inhibitors and substrates of NOS could have great therapeutic potential in the treatment of PCD patients. Currently, L-arginine supplementation has been studied in a variety of clinical situations in which the increase of NO production is desired. For example, digested L-arginine (31) and inhaled L-arginine (1) have been used in healthy subjects and patients with PCD to improve file bactericidal activity of the lungs, ciliary beating, and mucociliary beating.

Selective and more potent NOS inhibitors and NO donors, as well as noninvasive clinical methods with which to assess NO biochemistry will lead to a better understanding of its deleterious and beneficial effects, and to novel treatments for PCD patients.

In conclusion, our study has demonstrated that the levels of NO metabolites, such as N[O.sub.2.sup.-], N[O.sub.2.sup.-]/ N[O.sub.3.sup.-], and S-nitrosothiol, in exhaled breath condensate are not different from normal in patients with PCD, despite the marked decrease in exhaled NO levels. This may suggest that NOS activity is not decreased to such an extent as we expected on the basis of the detection of exhaled NO.

* From the Departments of Thoracic Medicine (Drs. Csoma, Balint Donnelly, Barnes, and Kharitonov) and Pediatric Respiratory Care (Drs. Bush and Wilson), Imperial College School of Medicine, National Heart and Lung Institute, London, UK. This study was supported by the European and Hungarian Respiratory Society the Hungarian Immunology and Allergology Society (Hungary), and the National Heart and Lung Institute (UK). Manuscript received June 4. 2002: revision accepted December 11, 2002.

Manuscript received June 4, 002; revision accepted December 11, 2002.

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Reproduction of this article is prohibited without written permission from the American College of Chest Physician (e-mail: persmissions@chestnet.org).

Correspondence to: Peter J. Barnes, DM, Department of Thoracic Medicine, Imperial College School of Medicine. National Heart and Lung Institute, Dovehouse St, London, SW3 6LY, United Kingdom; e-mail: p.j.barnes@ic.ac.uk

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