SUBJECTS

The study population consisted of 20 patients with stable COPD recruited from Servizio di Fisiopatologia Respiratoria, Azienda Ospedaliera di Parma (PR), Italy and 30 healthy control subjects. The subjects were divided into four groups based on their smoking habit (table 1). The groups were matched according to smoking history (22 of 30 subjects had a smoking history >30 pack years). Although normal individuals were not age matched, no relationship between age and exhaled NO has been reported, at least in adults.

Inclusion criteria for COPD were chosen according to the British Thoracic Society guidelines1: (a) evidence of airways obstruction (FEV₁ <80% of predicted and FEV₁/VC ratio <70%); (b) no improvement in FEV₁ of more than 15% after inhalation of 200 μg salbutamol; (c) history of chronic progressive symptoms such as wet cough, wheeze and breathlessness. Exclusion criteria were: (a) history of atopy or evidence of atopy on skin prick testing for common inhaled allergens, (b) history of asthma or other pulmonary disease, (c) treatment with oral or inhaled glucocorticoids or antibiotics during the month before entering the study. Patients were not excluded if they were receiving short acting or long acting β₂ agonists, or both, since these drugs are reported not to modify exhaled NO levels.11

The patients with COPD were clinically stable and had had no exacerbation of their disease, defined as increased dyspnoea associated with change in quality and quantity of sputum within the preceding month. Their smoking history was carefully controlled and a smoking history of at least 15 pack years was required. Before entry to the study patients were screened by clinical history, physical examination, electrocardiography, urinalysis, and haematological and blood chemistry tests. Chest radiography was performed to exclude other lung diseases.

All the subjects who smoked cigarettes at the time of the study, whether COPD patients or healthy controls, were considered to be current smokers; all the others were considered to be non-smokers or ex-smokers (smoking cessation >1 year). Current smokers in both groups were asked to refrain from smoking for at least 12 hours before the measurement of exhaled NO levels.

All the subjects gave their written informed consent to participate, and the study protocol was approved by the local ethical committee.

BREATHING MANOEUVRE

Subjects were seated and were asked to inspire room air via the mouth to total lung capacity and to exhale through a mouthpiece. Mixed exhaled air was collected using a three way stopcock into two sequential bags. In the first bag the first eight seconds (640 ml) of exhaled air was collected, representing the anatomical and mechanical dead space, and into the second bag the remaining exhaled air was collected. Subjects performed three exhalations and the mean value was calculated. In accordance with the European guidelines,12only the second portion of exhaled air was used for exhaled NO measurements.

The bags used were made of polyvinylfluoride film with a volume of 10 litres (Tedlar Samples Bags, SKC Inc, Philadelphia, USA); the concentration of NO in the bags did not change for at least six hours, as tested in separate experiments (data not shown).

During the expiration each subject maintained a constant expiratory flow (4.8 l/min) verified by a flow meter positioned between the mouth and the bags. Samples were discharged if the subject did not keep a constant flow. In a preliminary study we verified that an expiratory flow rate of 4.8 l/min with a constant resistance permitted a mouth pressure of 20 cm H₂O to be reached, as shown by a manometer (Dwyer Instrument Inc, Michigan City, Indiana, USA) with a side arm inserted between the mouth and the flow meter. With this mouth pressure the soft palate rises and closes the access to the nasal space above, preventing leakage of nasal NO into the mouth during expiration.12 To prevent condensation of vapour on the walls of the bags a Teflon column, 20 × 60 mm packed with 6 g of silica gel (particle size 1.7–4 mm; E Merck, Darmstadt, Germany) was inserted between the mouthpiece and the bags.

Samples of exhaled air packed in bags, covered with black cloths, were transported to the laboratory and analysed within 60 minutes. To measure ambient NO, samples of air from the room in which the subjects performed the test were collected into bags by a pump immediately before exhaled NO evaluations.

MEASUREMENT OF NO

The levels of exhaled and ambient NO were measured using a chemiluminescence NO analyser (Model 42S; Thermo Environmental Instruments Inc, Franklin, Massachusetts, USA). The analyser measures photons resulting from oxidisation of NO by ozone using a photomultiplier at a sampling flow rate of 0.7 l/min with a response time of 10 s (it takes three measurements to reach the highest stable exhaled NO value). The instrument was calibrated daily over the range of 0–100 parts per billion (ppb) with dilutions of a known NO source (SAPIO, Milan, Italy) by using a multigas precision flow calibrator (Dynamic Gas Calibration System, Model 146, Environmental Instruments Division, USA). Before all NO measurements were made, NO-free air was passed through the analyser for NO zeroing.

DATA ANALYSIS

Group data were expressed as mean (SE) or median and range where appropriate. Coefficient of variation was used to test the repeatability of exhaled NO measurements. Comparisons between data from COPD and control subjects were tested using the Mann-Whitney U test; correlations between exhaled NO levels and smoking history and lung function tests were tested with Spearman’s rank test. A p value of <0.05 was regarded as significant.