METHODS

Subjects and study design

A random sample of 15 healthy subjects, 20 steroid naïve patients with COPD, and 25 patients with COPD who were treated with inhaled corticosteroids participated in the cross sectional study (table 1).

Subjects attended on one occasion for clinical examination, spirometry, and EBC collection. The diagnosis of COPD was based on the Global Initiative for Obstructive Lung Disease (GOLD) guidelines.¹³ All patients had moderate COPD¹³ and were clinically stable with forced expiratory volume in 1 second (FEV₁) <80% predicted and FEV₁/forced vital capacity (FEV₁/FVC) ratio <70% which did not change markedly over 8 weeks. Patients with other respiratory or systemic diseases were excluded from the study. All study subjects were ex-smokers and had stopped smoking for at least 1 year. Smoking status was checked by measuring urinary cotinine levels which were <10 ng/ml in all study subjects. None of the steroid naïve COPD patients had received corticosteroids in the previous 4 weeks. Inhaled β adrenergic agonists and theophylline were also used in some patients (table 1).

Repeatability for eicosanoid measurements was assessed in 25 patients with COPD. A second EBC sample was collected within 7 days of the first sample.

The study was approved by the ethics committee of the Royal Brompton Hospital and Harefield Trust and informed consent was obtained from all subjects.

Pulmonary function

Spirometric tests were performed using a dry spirometer (Vitalograph Ltd, Buckingham, UK) and the best value of the three manoeuvres was expressed as an absolute value (litres) and as a percentage of the predicted value.

Measurement of exhaled eicosanoids

EBC samples were collected using a commercially available condensing chamber, as previously described (Ecoscreen; Jaeger, Hoechberg, Germany).⁸ Subjects breathed tidally through a mouthpiece connected to the condenser for 15 minutes.

LTB₄, LTE₄, PGF_2α,TxB₂, and PGD₂-methoxime (MOX), a stable derivative of PGD₂, were measured by enzyme immunoassays (Cayman Chemical, Ann Arbor, MI, USA). PGE₂ concentrations were measured by a radioimmunoassay (RIA).¹⁴

The intra-assay and inter-assay coefficients of variability of the eicosanoid assays were within 10% and 15%, respectively. LTB₄ and PGE₂ measurements in EBC were qualitatively validated by reverse phase high performance liquid chromatography.^15,16 The values of LTE₄, PGF_2α, and TxB₂ were referred to as eicosanoid like immunoreactivity (LI).

The possible influence of the ventilation rate on exhaled LTB₄, LTE₄-LI, and PGE₂ concentrations was excluded as previously described.¹² Saliva contamination of EBC was excluded by measuring amylase concentrations which were undetectable in all samples tested.

Statistical analysis

Eicosanoid concentrations in EBC were expressed as median values with the minimum to maximum range. A Kruskal-Wallis test followed by pairwise Mann-Whitney U tests was used to compare groups. Correlation was expressed as Spearman’s correlation coefficient. Significance was defined as a p value of <0.05. Within-subject repeatability of exhaled eicosanoid measurements was expressed as intraclass correlation coefficient.¹⁷

RESULTS

Exhaled LTB₄ was increased in both steroid naïve COPD patients (100.6 (73.5–145.0) pg/ml; p<0.001) and those treated with steroids (99.0 (57.9–170.5) pg/ml; p<0.001)] compared with age matched controls (38.1 (31.2–53.6) pg/ml; fig 1A). PGE₂ in EBC was also increased in both steroid naïve (98.0 (57.0–128.4) pg/ml; p<0.001) and steroid treated patients with COPD (93.6 (52.8–157.0) pg/ml; p<0.001) compared with healthy subjects (44.3 (30.2–52.1) pg/ml; fig 1B). There was no difference in exhaled LTB₄ (p=0.43) and PGE₂ (p=0.59) concentrations between steroid naïve and steroid treated patients with COPD. LTB₄ (p=0.86) and PGE₂ concentrations (p=0.57) were similar in patients with COPD treated or not treated with theophylline. There was no difference in LTB₄ (p=0.89) and PGE₂ concentrations (p=0.23) between COPD patients treated or not treated with β₂ agonists.

There was no difference (p=0.31) in LTE₄-LI between healthy subjects (15.5 (11.0–27.0) pg/ml), steroid naïve patients with COPD (23.3 (9.1–31.3) pg/ml), and steroid treated patients with COPD (19.5 (9.0–42.0) pg/ml). PGF_2α-LI was measurable in five steroid naïve COPD patients, in six steroid treated patients, and in five healthy subjects. In those subjects in whom PGF_2α-LI was measurable, its concentrations were higher in steroid naïve (15.0 (10.9–19) pg/ml; p<0.05) and in steroid treated patients with COPD (14.7 (10.0–21.7) pg/ml; p<0.05) than in healthy subjects (8.9 (5.9–10.9) pg/ml). PGD₂-MOX was measurable in six steroid naïve COPD patients (11.2 (8.7–15) pg/ml), in seven steroid treated COPD patients (11.4 (8.0–14.9) pg/ml), and in six healthy subjects (10.0 (8.0–15) pg/ml). In those subjects in whom PGD₂-MOX was measurable, there was no difference in the levels of this PG between the three study groups (p=0.80). TxB₂-LI was undetectable in any study subjects.

There was a correlation between LTB₄ and PGE₂ in both steroid naïve (r=0.75, p<0.001) (fig 2A) and steroid treated patients with COPD (r=0.67, p<0.001; fig 2B). The intraclass correlation coefficient was 0.72 for LTB₄, 0.82 for PGE₂, 0.68 for LTE₄-LI, 0.79 for PGD₂-MOX, and 0.73 for PGF_2α-LI. The intraclass correlation coefficient for TxB₂-LI was not calculated because this eicosanoid was undetectable in any study subjects.

DISCUSSION

We have identified the profile of exhaled LTs and PGs in COPD. Patients with COPD had about 2.5-fold higher LTB₄ concentrations in EBC than age matched control subjects, consistent with a primary pathophysiological role for LTB₄ in COPD.^18,19 LTB₄ concentrations in EBC were about 30-fold lower than those reported in sputum.²⁰ Exhaled PGE₂ was 2.2-fold higher in COPD patients than in healthy subjects. Since PGE₂ may have anti-inflammatory effects in the airways,^21,22 its increase, concomitant with enhanced levels of LTB₄, a potent pro-inflammatory mediator, may represent a mechanism to counteract lung inflammation in COPD. This hypothesis is further supported by the correlation found between LTB₄ and PGE₂ in both steroid naïve and steroid treated patients with COPD. PGF_2α-LI was detectable in the EBC in a few patients with COPD, and its concentrations were higher than those in healthy subjects. However, because of the small number of patients showing this pattern and the low concentrations detected, increased exhaled PGF_2α-LI in patients with COPD is likely to be of limited biological relevance.

The profile of exhaled eicosanoids in patients with COPD is different from that previously reported in patients with asthma.¹² Exhaled PGE₂ is selectively increased in patients with COPD, whereas LTE₄ is increased in patients with asthma,¹² but not with COPD. Exhaled LTB₄ is increased in both asthmatic and COPD patients, but the increase was more marked in the latter. We have previously shown that exhaled TxB₂-LI is measurable in about 50% of asthmatic patients,¹² whereas no TxB₂-LI was detectable in COPD patients in the present study. Identification of selective profiles of exhaled eicosanoids may help to differentiate between inflammatory lung diseases. The physicochemical properties of eicosanoids in EBC (volatile versus non-volatile) and their partition in EBC (aerosol particles versus water vapour) are largely unknown. The high inter-individual variability in the amount of aerosol particles in EBC indicates the need for a dilution marker.²³ However, the selective increase of structurally related compounds in the single subject (LTB₄ but not LTE₄, and PGE₂ but not PGD₂) is likely to reflect a real increase in the concentrations of these eicosanoids.

The effects of corticosteroids on eicosanoids in COPD in vivo are largely unknown. In our study there was no difference in exhaled LT and PG concentrations between steroid naïve and steroid treated patients with COPD. However, the cross sectional design cannot exclude steroid reduction of LTB₄ or PGE₂ and controlled studies with these drugs are required. Moreover, the effects of potential variables which may influence the effects of exhaled eicosanoids such as the use of previous medication, effects of feeding, effects of exercise, and diurnal variation need to be investigated.

In conclusion, we have shown that LTB₄ and PGE₂ are increased in EBC in patients with stable COPD. The profile of exhaled LTs and PGs in patients with COPD is different from that previously reported in patients with asthma. Identification of selective profiles of eicosanoids in EBC, a completely non-invasive method, may be relevant for the diagnosis and management of COPD.