The Role Of Nitrite In Exhaled Breath Condensate For Evaluating Asthma Severity

Background and objectivesBronchial asthma,or simply asthma,is a disease of chronic airway inflammation in which a variety of cells(such as eosinophils,mast cells,T cells,neutrophils,and airway epithelial cells) and cellular elements are involved.Asthma is characterized by Bronchial hyperresponsiveness(BHR) and its symptoms include dyspnea,wheezing, stridor,coughing,itching of the chest or an inability for physical exertion.Asthma always occurs and exacerbates at night and/or early morning,and will relieve spontaneously or with treatment.Asthma is a serious global health problem.People of all ages in countries throughout the world are affected by this chronic airway disorder that,when uncontrolled,can place severe limits on daily life and is sometimes fatal.The prevalence of asthma is increasing in most countries,especially among children. Asthma is a significant burden,not only in terms of health care costs but also of lost productivity and reduced participation in family life.Therefore,the research on asthma bears significant values of the public hygiene and social economy.The conventional observation of asthma was based on clinical symptoms and pulmonary function test,which did not reflect the pathophysiological features of asthma,the chronic airway inflammation.Currently,plenty of studies have demonstrated the significant association of exhaled nitric oxide(eNO) with airway inflammation and hypersensitivity,which indicated that the eNO could be used for the diagnosis of asthma and differential diagnosis of respiratory tract diseases. However,the detection of eNO requires the constant expiratory flow,and the device for its detection is expensive,so its clinical application is not widely available.EBC detection was a currently developed technique for the noninvasive detection of airway inflammatory state,it is a noninvasive modality evaluating the airway inflammation and oxidative stress and bears merits of simplicity,convenience,and good repetition, which is suitable for all kinds of patients including children and mechanical ventilated patients.Currently,some scholars proposed that EBC was a method for the eNO collection and the study of NO reaction products of EBC could reflect the changes of NO indirectly.The present study aimed to detect the content of NO₂^-(one of the NO reaction products) in EBC in asthmatic patients during acute episode,and compare with the normal control group to further investigate whether it can be used as an index for the evaluation of asthma severity.Subjects and methods2.1 Subjects:All the patients were consecutively from the emergency department or the department of respiratory medicine in the Sixth People's Hospital of Shenzhen.The 60 subjects enrolled were selected into our study.Patients were evaluated with diagnostic criteria of asthma prevention and treatment guideline in 2003 by asthma group of society of respiratory disease,CMA.The illness was graded according to the severity of the acute episode.There were 23 patients with mild asthma.10 cases were male and 13 cases were female.The average age was 40.0±5.0 years old(range 25～47 years old).There were 21 patients with moderate asthma.11 cases were male and 10 cases were female.The average age was 37.7±9.6 years old(range 20～45 years old).There were 16 patients with severe asthma.9 cases were male and 7 cases were female.The average age was 37.6±9.1 years old(range 19～49 years old).We selected 23 healthy normals.11 cases were male and 12 cases were female.The average age was 40.6±7.8 years old(range 18～48years old).There is no difference between groups when we compare their sex(χ² =0.720,P=0.868) and age(F=0.766,P=0.517).2.2 Exclusion criteria:patients with the age below 16 or above 50,those in the pregnant period and within one year after delivery were excluded.Smokers,or second-hand smokers in the recent three month regularity were excluded.Patients who eat any pickle in recent 3 days were excluded.Patients with infectious diseases in respiratory system four weeks before the trial were excluded.Those with acute episode complicated with infection in respiratory system were excluded.And those with chronic respiratory diseases besides asthma were also excluded.Those with hypertension and diabetes mellitus were excluded.Those on corticosteroids(inhaled, orally,intraveneously),theophiline,bronchodilators,leukotrienes regulators and sodium cromoglycate 72 h before trial were excluded.2.3 Methods2.3.1 Pulmonary function test:pulmonary function meter was from Jaeger Company, after EBC collection,mean expiratory flow in 50%of the vital capacity(MEF 50%) was detected,as well as other pulmonary function indices such as FEVI%.2.3.2 EBC collection:the EBC collector(Rtube) from Respiratory Research Company,USA was used.The device was comprised of refrigerating aluminum tube, insulation cover,collecting tube,push rod and lid.The collecting tube had two movable flaps,during inspiration,air flow entered from the bottom flap and during expiration the flow came through another flap and entered the condensation chamber on the top of the device to form condensation fluid.The patient gargled with distilled water three times before EBC collection.The refrigerating aluminum tube was taken out from -10℃refrigerator to assemble the EBC collector.The subject was in sedentary position and tidal-breathed quietly by mouth.The duration of collection was 15 min.Swallow of salivary was constantly done,once the cough or belching occurred,the interface was removed to prevent pollution by salivary and Gastric contents.2.3.3 Detection of NO₂^- colored substance was produced by NO₂^- and chromogenic reagent through Griess reaction,and then the absorbance of NO₂^- was detected with spectrophotometry.The concentration of NO₂^- was obtained with standard curve method,whose detailed procedure was as follows,NO₂^- solutions with different concentrations were prepared and their respective absorbance were measured,then the standard curve was made.Under the same condition,the absorbance of the sample solution was detected,and its concentration was read from the standard curve.2.3.4 Collection and treatment of the induced sputum and its cytological detection All the subjects inhaled 200μg salbutamol aerosol and nebulized 3% sodium chloride solution consecutively after pulmonary function test and EBC collection.If there was sputum,the subject wiped their nose,gargled,drunk water and coughed sputum.If there was no sputum,the nebulization was continued with 4% or 5%sodium chloride solution until 3-5 ml sputum sample was collected.If the subject could not tolerate the sputum inducement procedure,the procedure should be discontinued and relevant treatment was initiated until the symptoms were relieved. The sputum smear showed plenty of columnar epithelial cells and phagocytes,and the sputum specimen with the ratio of squamous epithelial cells to nucleated cells less than 20%was regarded as qualified.The sputum component in the specimen was selected with blunt-head forceps,which was then mixed with four times volume of 0.1%dithiothreitol solution and incubated at 37℃for 15 min,then,the sample was filtered with 48μm gauze,the infiltrates was centrifugated at 2000 r/min for 10 min. The cells was settled by re-suspension with PBS(pH7.4,0.1 mmol / L),followed by centrifugation at 2 500 r/min,the cell sediment was smeared.Three or more than three smears were prepared for each specimen,and processed with Wright stain,then 400 non-squamous cells were counted under oil lens,the percentage of eosinophil (Eos%) was calculated.Specimen with squamous cells more than 20%was regarded as disqualified.2.4 Statistical analysisData was analyzed using a software package(SPSS 15.0).Numerical data were expressed as frequency.Chi-Square test was applied in comparison of numerical data. Measurement data were expressed as mean±standard deviation.We used Levene test to analysis weather the measurement data was homogeneity.Comparison with two means were analysed through t-test.One-way ANOVA(equal variance assumed)or corrected one-way ANOVA((Welch) equal variance not assumed) was used to compare multiple means,and LSD-t test(equal variance assumed) or Tambane's T2 test(equal variance not assumed) was used to analyze the groups between them.The correlation of two variables was analysed by Pearson rank correlation coefficients.Significant difference was defined as P＜0.05.Results3.1 Comparison of NO₂^- in EBC,Eos%in induced sputum and MEF50%, FEV1%in asthmatic and control groupsNO₂^-(2.33±1.12)μmol/L in asthmatic group was significantly higher than that of control group(0.59±0.25)μmol/L(t=-11.301,P=0.000).Eos%in induced sputum in asthmatic group was(23.43±8.47)%,Eos%in induced sputum in normal group was(1.36±0.37)%,there was significantly difference between them (t=-20.121,P=0.000).The MEF50%and FEV1%in the asthmatic group were (62.99±15.56)%,(68.55±16.85)%respectively,which were significantly lower than corresponding values of(91.17±9.28)%and(93.40±8.49)%in the control group (t=8.128,P=0.000;t=8.862,P=0.000).3.2 Comparisons of EBC NO₂^-,Eos%in induced sputum and MEF50%, FEV1%in asthmatic patients on acute episode with various severities and healthy control.The EBC NO₂^- in the mild,moderate,severe acute episode groups and the control group were(1.42±0.25)μmol/L,(2.24±0.66)μmol/L,(3.77±0.88)μmol/L, (0.59±0.25)μmol/L respectively,and the multiple comparison was statistically significant(F=107.794,P=0.000).Eos%in induced sputum in the mild,moderate, severe acute episode groups and the control group were(16.36±3.00)%,(22.86±4.74) %,(34.33±5.95)%,(1.36±0.37)%respectively,and the multiple comparison was statistically significant(F=468.143,P=0.000).MEF50%in the mild,moderate,severe acute episode groups and the control group were(75.48±11.20)%,(61.99±9.97)%,(46.33±10.00)%,(91.17±9.28)%,respectively,and the multiple comparison was statistically significant(F=68.712,P=0.000).FEVI%in the mild, moderate,severe acute episode groups and the control group were(82.70±6.52)%, (70.67±7.78)%,(45.44±9.52)%,(93.40±8.49)%respectively,and the multiple comparison was statistically significant(F=121.638,P=0.000).Moreover,the more severe the episode,the higher the value of NO₂^- and Eos%,the lower the MEF50% and FEV1%3.3 The association analysis of EBC NO₂^-,Eos%in induced sputum and pulmonary function indices in asthmatic patients on acute episodeEBC NO₂^- level was in positive association with Eos%in induced sputum (r=0.762,P=0.000).The concentration of exhaled nitrite correlated negatively with MEF50%(r=-0.830,P=0.000)and with FEV1%(r=-0.724,P=0.000).Conclusions1.NO₂^- levels in expiratory condensation during acute episode with various severity (mild,moderate,severe) were significantly different(F=68.123,P=0.000).2.NO₂^- in EBC of asthmatic patients can be used as an index for the evaluation of severity of the acute episode.