Respiratory Treatment and Prevention (Advances in Experimental Medicine and Biology)

twice. The first sample was obtained in the morn-
ing after at least 6 h of refraining from smoking
during nighttime. The second sample was col-
lected 60 min after smoking a single cigarette of
a commercial brand with a tar content of 6 mg
and nicotine content of 0.5 mg. Both samples
were analyzed for IL-1β, TNF-α, and MDA.

2.2 Study Participants

Nineteen consecutive active smokers with sta-
ble COPD who agreed to participate in the
study during a routine follow-up visit in the
outpatient clinic of the Department of Internal
Medicine, Pneumology and Allergology of
Medical University of Warsaw in Poland were
enrolled into the study. The inclusion criteria
were as follows: 1/diagnosis of COPD in accor-
dance with the recommendations of the Global
Initiative for Obstructive Lung Disease (GOLD
2015 ); 2/post-bronchodilator forced expiratory
volume in one second (FEV 1 )50 % of
predicted value; 3/current smoking; 4/lack of
exacerbation or respiratory infection within
4 weeks before inclusion; and 5/no systemic
or inhaled steroid use 6 weeks prior to study
onset.
The second investigated group consisted of
19 asymptomatic young (<25 years of age) cur-
rent smokers with a negative history of respira-
tory diseases and normal spirometry. Subjects
who had a respiratory infection within 4 weeks
before the study onset were excluded.
In all patients lung function was assessed by
spirometry (Lungtest 1000, MES, Cracow,
Poland), which was performed in accordance
with the recommendations of the European
Respiratory Society and the American Thoracic
Society (Miller et al. 2005 ; Pellegrino
et al. 2005 ) after refraining from smoking for at
least 6 hours before the first EBC collection. In
COPD patients, spirometry was performed after
administration of their routine inhaled
medication.

2.3 Exhaled Breath Condensate

(EBC) Collection

and the Evaluation

of Biomarkers

EBC was collected and processed according to

the ATS/ERS recommendations (Horvath

et al. 2005 ) with the use of the TURBO-

DECCS 09 system (Medivac, Parma, Italy) dur-

ing tidal breathing for 20 min and 5 C conden-

sation temperature. The obtained samples were

portioned in 250μL aliquots and immediately

stored at 70 C for subsequent analysis.

The concentration of MDA, IL-1β, and TNF-α

in EBC was measured by ELISA method. The

following commercial ELISA kits were used:

CLIA Kit for general malondialdehyde (EIAab;

Wuhan, China, Catalog No EO597Ge), Human

IL-1 beta ELISA kit (RayBiotech; Norcross, GA,

Catalog No: ELH-IL1b), QuantikineHS ELISA

Human TNF-alpha immunoassay (R&D;

Minneapolis, MN, Catalog NoHSTA00D). The

sensitivity of the applied kits was 0.15 nmol/mL

for MDA, 0.3 pg/mL for IL-1β, and 0.191 pg/mL

for TNF-α, respectively. EBC samples for IL-1β

and TNF-αmeasurements were undiluted, while

MDA levels were evaluated in twice diluted con-

densate samples. None of the samples were

repeatedly thawed/frozen for analysis. To

achieve maximal reproducibility and reliability

of our measurements the samples were not

lyophilized.

2.4 Statistical Analysis

Data are presented as median and interquartile

range (IQR). The differences between continu-

ous variables in unrelated groups were tested

with the use the non-parametric Mann–Whitney

U test. Spearman’s rank correlation coefficient

was applied to test potential correlations between

different continuous variables. The differences

between the related samples (repeated

measurements) were tested with non-parametric

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