- Email: [email protected]
Leukotriene E4 in urine in patients with asthma and COPD—The effect of smoking habit
ARTICLE IN PRESS Respiratory Medicine (2007) 101, 826–832
Leukotriene E4 in urine in patients with asthma and COPD—The effect of smoking habit E. Gakia, G. Papatheodoroub, E. Ischakia, V. Grammenoua, I. Papaa, S. Loukidesc, a
Pneumonology Department, Athens Veterans Hospital, Athens, Greece Clinical Research Unit, Athens Army General Hospital, Athens, Greece c University of Athens Medical School, Smolika 2, Voula, Athens 16673, Greece b
Received 9 April 2006; accepted 28 June 2006
KEYWORDS Asthma; Chronic obstructive pulmonary disease; Leukotriene E4; Smoking; Eosinophils
Summary Leukotriene E4 (LTE4) is implicated in asthma pathophysiology and possibly in chronic obstructive pulmonary disease (COPD) as one of the causes of persistent bronchoconstriction and mucus hypersecretion. Cigarette smoking stimulates cysteinyl leukotrienes (CysLTs) production. We investigated whether LTE4 is equally increased in asthma and COPD and whether smoking significantly affects LTE4 levels. Secondary outcomes involved correlations with inflammatory and functional parameters. We studied 40 patients with COPD [20 smokers], 40 asthmatics [20 smokers] and 30 healthy subjects [15 smokers]. Spirometry (FEV1% pred., FEV1/FVC) was performed, urine was collected for measurement of LTE4 and creatinine, induced sputum was collected for differential cell counts and serum for ECP. LTE4/creatinine levels (pg/mg) [mean (SD)] were increased in asthmatic patients compared to COPD and controls, [125.6(54.5) vs. 54.5(19) vs. 55.9(18.9) pg/mg, respectively, Po0.0001 for asthma]. Smoking significantly affects LTE4 levels only in asthmatic patients [164 (48) vs. 87 (26.3), Po0.0001 for smokers]. The only significant correlation was between eosinophils in induced sputum and LTE4/creatinine levels in asthmatics. In conclusion, patients with asthma presented higher LTE4 values compared to normals and patients with COPD. Smoking significantly affects LTE4 values only in asthmatics indicating a different underlying CysLTs inflammatory process in this condition. & 2006 Elsevier Ltd. All rights reserved.
Introduction Corresponding author. Tel.: +30 210 8954603;
fax: +30 210 7770423. E-mail address: [email protected] (S. Loukides). 0954-6111/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2006.06.031
Cysteinyl leukotrienes (CysLTs) are a group of inflammatory mediators, which has received interest in the recent years. CysLTs (leukotrienes C4, D4 and E4) are central mediators in
ARTICLE IN PRESS Leukotriene E4 in urine in patients with asthma and COPD asthma pathophysiology. Several inflammatory cells have the biosynthetic capacity to produce CysLTs and Leukotriene E4 (LTE4) is a stable end product of CysLTs metabolism in the human lung. Urine LTE4 is a reliable marker of endogenous CysLTs formation and measurement of LTE4 offers a possibility to monitor changes in the rate of CysLTs production.1 Cigarette smoking stimulates CysLTs production in animal models but also in humans.2 The role of CysLTs in chronic obstructive pulmonary disease (COPD) remains uncertain despite previous but limited evidence which indicates increased production in different fluids in this disorder.3,4 However, there is evidence that CysLTs increase microvascular permeability, evoke mucus hypersecretion and are potent smooth muscle constriction agents,5 features that are present in the airways of COPD patients, and this suggests that they might play a role in this disorder. We tested the hypothesis that LTE4 values are equally increased in both asthma and COPD patients, and smoking habit significantly affects the above measurements. Therefore, we assessed the levels of LTE4 in the urine of patients with COPD and asthma, as well as in healthy controls, in an attempt to clarify the in vivo role of this mediator. Smoking as a possible factor that might affect LTE4 values was also tested after subdividing the study groups according to their smoking habit. Additionally, we investigated whether airway or systemic eosinophilic inflammation is implicated in the pathophysiology of this mediator in the two diseases, by assessing sputum differential cell counts and serum eosinophilic cationic protein (ECP) levels. Finally, we examined whether the functional status, as assessed by forced expiratory volume in 1 s (FEV1%) predicted and the FEV1/ FVC (FVC—forced vital capacity) ratio, is implicated in the measurements of LTE4.
Materials and methods Subjects Subjects’ characteristics are summarized in Table 1. The diagnosis of COPD was established according to the GOLD Guidelines,6 whereas the GINA Guidelines were used for the diagnosis of asthma.7 All subjects in the three study groups were subdivided according to their smoking habit (for COPD Table 1
827 patients this subdivision referred to current and exsmokers). A total of 145 subjects were initially recruited in order to achieve the final number of patients. Forty COPD patients (20 current smokers, all non-atopic and steroid-naı¨ve) were recruited. All COPD patients were characterized by the absence of eosinophils in induced sputum. All patients were receiving occasionally short acting b2-agonists as relief medication. Ex-smokers with COPD stopped smoking at least 2 years before entering the study. Forty atopic asthmatics, matched for age, BMI and lung function with COPD patients (20 current smokers, 20 never smokers, matched for age, BMI, lung function and smoking habit with the COPD patients who were current smokers; all steroid-naı¨ve), were also studied. All asthmatic patients had positive bronchodilation tests (increase of pre-bronchodilator FEV1412% after administration of salbutamol at dose 400 mcg by meter dose inhaler). They were occasionally receiving short-acting b2-agonists as relief medication. Thirty normal subjects (15 current smokers, 15 never smokers, matched for the smoking habit with the respective asthmatic and COPD smokers), with no history of any disease, served as controls. All were non-atopic, had normal lung function and negative bronchial challenge testing, and none of them was receiving any kind of medication at the time of the study. Normal subjects were younger compared to asthma and COPD patients since it is difficult to find healthy subjects without any comorbidities in the age of 65 years old. All patients were clinically stable, i.e. they had no evidence of acute exacerbation for at least 4 weeks prior to the study. None of our patients was receiving any antiinflammatory treatment (including corticosteroids, longacting b2 agonists, or leukotriene receptor antagonists), theophylline, inhaled or oral mucolytics, or long-term oxygen therapy. Atopy was defined by the high values of total IgE and on the basis of positive skin tests to six common aeroallergens.
Study protocol details Lung function measurements were performed in all subjects one day before the urine and blood collection and sputum induction. In all subjects, the collection of urine and blood
Subject characteristics.
Age (years) BMI (kg/m2) FEV1 (% pred) FEV1/FVC ratio (%) Smoking habit (pack-years) ECP Eosinophils (%) Neutrophils (%)
Controls (n ¼ 30) 9F–21M
Asthma (n ¼ 40) 14F–26M
COPD (n ¼ 40) 12F–28M
48 (8.5) (33–67) 26 (3.5) (20–35) 99 (10.5) (85–123) 99.5 (9) (89–125) n ¼ 15 38 (9) (19–80) 6(2) (3–9) ND ND
63 (14) (31–80) 28.4 (4) (20–40) 71 (16) (42–98) 68 (4) (59–76) n ¼ 20 37.5 (10) (20–55) 14.5(7) (5–35) 7.5 (4) (2–17) 28.5 (6) (22–40)
64 (8) (49–81) 27(4) (21–34) 67 (9) (49–85) 64 (3) (57–70) n ¼ 20 41 (11) (25–63) 6.5(2) (3–10.5) 0 64(7) (45–73)
Data are presented as mean (SD) with ranges in parenthesis. ND ¼ not done, M ¼ male, F ¼ female. Smoking habit refers only to the subgroups of current smokers in each group.
ARTICLE IN PRESS 828
E. Gaki et al.
preceded the sputum induction procedure, in order to avoid any effect of sputum induction procedure on the urine and blood measurements. All subjects were asked not to smoke 2 h before each procedure. In order to ensure the subjects’ compliance to this recommendation, they were asked to stay in our laboratory for 2 h under the supervision of one of the investigators. Sputum induction was not performed in normal subjects. Metacholine test was performed only in normal subjects on a separate day of the protocol. All subjects were asked not to receive short-acting bronchodilators at least 12 h before the collection of samples and the lung function tests. LTE4/creatinine values were compared among patients with COPD, patients with asthma and normal subjects. Similar comparisons were performed in the study subgroups. Correlations between LTE4/creatinine values and study parameters were also performed in all study groups. All subjects were recruited from the outpatient clinics of the Athens Veterans Hospital. This is a general hospital that serves veterans and civilians. The Scientific Committee of the hospital approved the study protocol, and all participants gave written informed consent.
immunoassay (EIA) (Cayman Chemical; Ann Arbor, MI, USA, No. 520411). The detection limit of the assay was 25 pg/mL. The results were normalized to the creatinine concentration determined in the same sample and the LTE4/creatinine ratios were used for subsequent studies. All urine samples were tested for protein, bilirubin, ketone, urobilinogen, glucose and hemoglobin and if any of these substances were presented the sample was excluded from the study. According to manufacturer’s guidelines and in order to check the need of purification, all samples diluted 1:3 with distilled water. LTE4 was measured in diluted and undiluted samples. The two different dilutions of the samples showed differences less than 18% in the final calculated LTE4 concentration, so the samples were measured without purification.
Metacholine challenge
ECP measurement
Bronchial hyperresponsiveness to metacholine was measured and performed by a rapid metacholine inhalation test for the determination of PD20, as previously described for histamine.8 PD20 was determined by linear interpolation on a semi-logarithmic scale. The test was performed according to the instructions established by the American Thoracic Society guidelines.9
The concentrations of ECP in the serum were determined by a specific enzyme immuno-assay (Pharmacia Diagnostics AB; Uppsala, Sweden) in an auto analyzer (Uni CAP 100) with a lower limit of detection of 2.0 mg/L.
Lung function Pulmonary function tests were measured with a dry spirometer (Vica-test, Model VEP2; Mijnhardt; Rotterdam, Holland). FEV1 and FVC were measured according to the American Thoracic Society guidelines.10
Creatinine measurement Creatinine concentration in urine was measured with a kinetic method. The complex formed by creatinine and picric acid (8.8 mmol/L) in an alkaline medium (NaOH 0.4 M) was measured for 1 min at 492 nm.
LTE4/creatinine repeatability measurements Repeatability of LTE4/creatinine measurements was evaluated in 20 subjects [4 controls (2 smokers),8 COPD patients (4 from each subgroup) and 8 asthmatics (4 smokers)]. Repeatability of LTE4/creatinine measurements was tested in samples from the same subjects collected on two consecutive days.
Statistical analysis Sputum induction and processing Sputum was induced as previously described.11 Subjects were asked to blow their noses, rinse their mouths, and swallow the water in the aerosol to minimize contamination with postnasal drip and saliva. An induction procedure using inhalation of an aerosol of hypertonic saline solution (3.5%) generated by a DeVilbiss ultrasonic nebulizer (2696 Somerset PA, USA) was chosen. Induction was performed for periods of 30 s, and 1, 2, 4 and 8 min. The patients were asked to expectorate sputum after the 4 and 8 min period. At least 2 mL of sputum were collected in a sterile container. The person who did the differential cell counts in sputum (G.P.) was not aware of the clinical and functional status of the patients or the results of bronchial challenge testing.
LTE4 measurements LTE4 concentrations were measured in a non-purified urine morning sample with a specific competitive enzyme
Data are expressed as mean (SD), unless otherwise mentioned. Values among three or more groups were evaluated using one-way analysis of variance (ANOVA) with an appropriate post hoc test for multiple comparisons (Bonferroni). For comparisons between two groups, Mann–Whitney U tests were used. Spearman’s correlation coefficient was used to investigate correlations between parameters. Repeatability of LTE4/creatinine measurements was assessed with the Bland and Altman test.12 A P-value o0.05 was considered significant.
Results Subjects characteristics in induced sputum Asthmatic patients were mainly characterized by a predominant eosinophilia in induced sputum whereas COPD patients were characterized by a predominance of neutrophilic inflammation (Table 1). Asthmatic smokers presented
ARTICLE IN PRESS
Ex-smokers (n ¼ 20) 5F–15M
with significantly higher values of % eosinophils in induced sputum compared to non-smokers (Table 2, Po0.003). Asthmatic smokers presented a trend for higher values of % neutrophils in induced sputum compared to non-smokers, that was marginally statistically significant (Table 2, P ¼ 0.048). No significant differences were observed between current and ex-smokers patients with COPD in regard to % neutrophils in induced sputum (Table 2, P ¼ 0.79)
The measurements of LTE4/creatinine on 2 consecutive days presented good repeatability. LTE4/creatinine on days 1 and 2 were 80.96(52) pg/mg and 82.04(52) pg/mg, respectively. The correlation between LTE4/creatinine measurements on two consecutive days was significant (r ¼ 0.94, Po0.0001). The mean difference with limits of agreement was 1.0874.9 (mean72 SD) in the Bland and Altman plot.
Discussion In this prospective cross-sectional study we have found significant elevation of LTE4 levels in the urine of patients
13.5 (5) (5.5–23) 4.5 (2) (2–9) 25 (6.5) (22–35)
65(6) (49–73) 27 (4) (21–33) 66 (8.5) (49–81) 64 (3) (57–70) 41 (11) (25–63) 6 (2) (4–9) 0 66(8) (45–73) 62 (16.5) (31–79) 27.5(4) (20–34.5) 72 (17) (42–98) 69 (4.5) (60–76)
5 (2) (3–9) ND ND
65 (11) (47–80) 29(4) (24–40) 70.5 (15) (53–97) 68(4) (59–72) 37.5 (10) (20–55)) 16 (9) (5–35) 10 (4)** (4–17) 31.5 (6) (28–40) 46 (7) (34–59) 27(3) (21–35) 100(11) (85–123) 102.5 (10) (89–125)
Smokers (n ¼ 20) 7F–13M Smokers (n ¼ 15) 4F–11M
Non-smokers (n ¼ 15) 5F–10M
Asthma
Repeatability of LTE4/creatinine measurements
Table 2
Major correlation data are summarized in Table 3. Briefly, the values of LTE4/creatinine presented a significant correlation with eosinophils in induced sputum in asthmatics (r ¼ 0.83, Po0.0001). This significant correlation was also observed in smoking asthmatics, but was not expressed in asthmatic non-smokers (Table 3, Figs. 3A and B). No correlation with ECP levels was found. No significant correlation was observed between the levels of LTE4/ creatinine and any of the parameters tested in COPD patients. No significant correlation was observed between LTE4 and the degree of lung function impairment or the smoking habit in pack-years in all study groups. Neutrophils in COPD patients did not present any significant correlations with LTE4/creatinine (r ¼ 0.02, P ¼ 0.77).
Subject characteristics according to smoking habit.
Correlations
Controls
The levels of LTE4/creatinine (pg/mg) were increased in asthmatic smokers compared to non-smokers [164 (48) vs. 87 (26.3) pg/mg, Po0.0001, Fig. 2]. No significant difference was observed between smokers and non-smokers in the COPD and control groups [58.3 (23) vs. 50.5(13) pg/mg, P ¼ 0.4, 56(20) vs. 56 (18), P ¼ 0.83, respectively, Fig. 2]. However, a trend for higher values in COPD smokers compared to ex-smokers was evident.
49.5 (9.5) (33–67) 26(4) (20–35) 98 (10.5) (87–123) 96.5 (6) (89–106) 38 (9) (19–80) 6.7 (1.5) (4.5–9) ND ND
Non smokers (n ¼ 20) 7F–13M
Effect of smoking
Age (years) BMI (kg/ m2) FEV1 (% pred) FEV1/FVC ratio (%) Smoking habit (pack–years) ECP Eosinophils (% ) Neutrophils (%)
COPD
The levels of LTE4/creatinine (pg/mg) were increased in asthmatic subjects compared to COPD and controls [125.6(54.5) vs. 54.5(19) vs. 55.9(18.9) pg/mg, respectively, Po0.0001 for asthma, Fig. 1]. No statistically significant difference was found between COPD patients and normal subjects [54.5(19) vs. 55.9(18.9) pg/mg, P ¼ 0.3, Fig. 1].
Smokers (n ¼ 20) 7F–13M
LTE4/creatinine levels in major study groups
Data are presented as mean (SD) with ranges in parenthesis. ND ¼ not done, M ¼ male, F ¼ female, *ex-smoking history, **Significant higher compared to non-smoking asthmatics, P ¼ 0.003, Non-smokers ¼ never smokers.
829
62 (7.5) (51–81) 26.5(4) (22–34) 68 (8) (52–85) 65(4) (58–70) 42 (9)* (32–60) 6.5(2) (3–10.5) 0 62 (5.5) (48–70)
Leukotriene E4 in urine in patients with asthma and COPD
ARTICLE IN PRESS 830
E. Gaki et al. p<0.0001
LTE4 / creatinine pg/mg
300 250 200 150 100 50 0 Normals
Asthma
COPD
Figure 1 LTE4/creatinine concentration in control subjects [all n ¼ 30 ’], patients with asthma [all n ¼ 40 m, and patients with COPD [all n ¼ 40 ., Each symbol represents one individual. Horizontal bars represent mean values. See text for details.
LTE4/creatinine pg/mg
300
p<0.0001
250 200
p=0.83 p=0.4
150 100 50 0 NS
S
Normals
NS
S
Asthma
ES
S
COPD
Figure 2 LTE4/creatinine concentration in non-smoking control subjects [n ¼ 15 &], smoking control subjects [n ¼ 15 ’], non-smoking patients with asthma [n ¼ 20 ,], smoking patients with asthma [n ¼ 20m], ex-smokers with COPD [n ¼ 20 J] and smokers with COPD [n ¼ 20 K]. Each symbol represents one individual. Horizontal bars represent mean values. S ¼ smoking, NS ¼ non-smoking, ES ¼ Ex-smoking. See text for details.
Table 3
with asthma compared to matched COPD patients and healthy controls. Smoking habit significantly affects LTE4 levels only in asthmatic subjects. The values of LTE4 in urine present significant correlations with eosinophils in induced sputum only in asthmatics; interestingly, the absence of smoking habit seems to affect negatively the above significance. The measurements of LTE4/creatinine on 2 consecutive days are repeatable. CysLTs are synthesized from arachidonic acid through a 5-lipoxygenase pathway and are mainly generated by many inflammatory cells, particularly eosinophils, mast cells and macrophages. The implication of LTE4 in asthmatic inflammation is well established, and the increased levels of LTE4 in the urine of asthmatic subjects reported in this study are comparable to those published by previous investigators.13,14 However, this is the first study to our knowledge that evaluates the levels of LTE4 in urine of patients with COPD. Two previous studies have reported increased CysLTs levels in patients with COPD; however, in the first study the mediator that was used (LTC4) is not considered as a stable end product in CysLT production,2 and in the second the biological fluids studied did not represent sources which would definitely express airway inflammation.15 Interestingly, the levels measured were not significantly higher compared to normal subjects, indicating that the 5-lipoxygenase pathway of arachidonic acid metabolism may not be activated in COPD patients. The reason for this observation might be attributed to the capability and/or the activation state of the inflammatory cells which participate in the CysLTs production. This is partially supported by the findings of our study that show a positive correlation between sputum eosinophils and LTE4 values in asthma and the absence of any correlation between induced sputum cells and LTE4 values in COPD. However, this positive correlation might also express the CysLTs-induced prolonged survival of eosinophils in the airways that contributes to the maintenance of the inflammatory process in asthma.16 The absence of similar correlation with ECP might be related to the low sensitivity of this mediator for the assessment of eosinophilic inflammation. A previous observation, where the use of a CysLT1 receptor antagonist led to improvement of some COPD patients with less fixed airflow obstruction was not tested in this study, since the methodology used did not include a proper
Correlations of LTE4 in COPD and asthmatic patients as well as in asthmatic smokers and non-smokers.
LTE4 Asthma
FEV1 (% predicted) FEV1/FVC ratio Eosinophils (%) Smoking (py) ECP (mg/L) Neutrophils
COPD
Asthma smokers
Asthma non-smokers
r
P-value
r
P-value
r
P-value
r
P-value
0.02 014 0.83 ND 0.10 0.03
0.80 0.35 o0.0001 ND 0.50 0.86
0.04 0.21 ND ND 0.002 0.02
0.74 0.10 ND ND 0.83 0.77
0.07 0.1 0.77 0.24 0.21 0.04
0.74 0.63 o0.0001 0.25 0.73 0.81
0.10 0.07 0.31 ND 0.08 0.06
0.6 0.77 0.10 ND 0.71 0.88
Bold letters indicate significant correlations. ND ¼ not done.
ARTICLE IN PRESS Leukotriene E4 in urine in patients with asthma and COPD
r=0.77 p<0.0001
16 14 12 10 8 6
10 % eosinophilsin induced sputum in asthmatic non smokers
% eosinophils in induced sputum in asthmatics smokers
18
831
8 6 4 2
4 100 (A)
r=0.31 p=0.1
125 150 175 200 225 250 LTE4/creatinine pg/mg in asthmatics smokers
40 (B)
60 80 100 120 140 160 LTE4/creatinine pg/mg in asthmatics non smokers
Figure 3 Correlations between the concentration of LTE4/creatinine and eosinophils (A) in asthmatic smokers (r ¼ 0.77, Po0.0001) and (B) in asthmatic non-smokers (r ¼ 0.35, P ¼ 0.1).
placebo-controlled design; thus, any conclusions arising were not strong enough to confirm a potential role of CysLT1 receptor antagonists in COPD.17 Smoking affects significantly LTE4 levels in asthma but not in COPD and normal subjects. The positive effect of smoking in CysLTs production in asthma, irrespective of the degree of smoking habit, might be related to the number and/or the activation state of cells responsible for producing CysLTs. Cells like alveolar macrophages and mast cells, which are activated in asthma and also increased by smoking, might stimulate the production of CysLTs via the preexisting activated 5-lypoxygenase pathway. This theory is supported by previous observations where in vivo data showed the protective effect of a leukotriene receptor antagonist on cigarette smoking-induced lung injury.18 Similar findings regarding the concentration of the eicosanoid PGE2 in smoking asthmatics19 indicated that eicosanoid metabolism in asthmatics is significantly influenced by smoking. Data from the present study showing absence of significant correlation between eosinophils in induced sputum and the levels of LTE4 in the urine of non-smoking asthmatics may represent further evidence in this direction. However a possible limitation of our study by combining the above observations might be that they just represent the prolonged survival of eosinophils that is induced by the increased CysLTs or the eosinophil-induced CysLTs production. The above observation about the effect of smoking on asthmatics might have practical implications, since it might partially explain the difference in the response to inhaled steroid treatment in smoking asthmatics, once the CysLTsrelated pathophysiology is not sensitive to the treatment with steroids.20,21 Interestingly, the above observations were not present in normal subjects and COPD patients. This finding does not seem reasonable, but it could be attributed to the previously inactivated 5-lipoxygenase pathway. In normal subjects it might also be explained by previous observations where the increase in LTE4 was attributed to acute effects rather than to indirect effects resulting from chronic smoking exposure, since 5-lipoxygenase is not in a proper state to be activated and to produce CysLTs.2 However, some previous data supports that smoking in a dose-dependent manner significantly affects LTE4 values in normal subjects.22
The repeatability data presented in this study are similar to data reported for COPD using another biological fluid,23 indicating that, in stable patients with chronic inflammatory airway diseases and persistent inflammation, the measurement of mediators involved in the inflammatory process may be repeatable. This study presents one possible limitation, regarding the measurement of LTE4 in urine, which was performed by an EIA without purification. However, LTE4 has been shown to be stable in urine samples stored at 20 1C for months without the addition of preservatives. Additionally, LTE4 levels in crude urine samples assessed by EIA have been shown to present significant correlation with LTE4 measurements performed after purification on solid-phase extraction followed by separation on reversed-phase highperformance liquid chromatography24 and finally a specific procedure assessing interference in diluted and undiluted samples as suggested by the manufacturer was performed. In summary, we report that patients with asthma present higher urine LTE4 values compared to patients with COPD and to healthy controls. Smoking in asthmatics seems to be a critical parameter that might influence the above values. Eosinophils as a cellular source or as a result of prolonged survival are probably the key inflammatory cells in CysLTsrelated inflammatory process in smoking asthmatics.
References 1. Wennergren G. Inflammatory mediators in blood and urine. Paediatr Respir Rev 2000;1:259–65. 2. Saareks V, Riutta A, Alanko J, Ylitalo P, Parviainen M, Mucha I, et al. Clinical pharmacology of eicosanoids nicotine induced changes in man. J Physiol Pharmacol 2000;51:631–42. 3. Mitsunobu F, Mifune T, Hosaki Y, Ashida K, Tsugeno H, Okamoto M, et al. Enhanced production of leukotrienes by peripheral leukocytes and specific igE antibodies in patients with COPD. J Allergy Clin Immunol 2001;107:492–8. 4. Piperno D, Pacheco Y, Hosni R, Moliere P, Gharib C, Lagarde M, et al. Increased plasma levels of atrial natriuretic factor, renin activity and leukotriene C4 in chronic obstructive pulmonary disease. Chest 1993;104:454–9. 5. Dahlen SE. Pharmacological characterization of leukotrienes receptors. Am J Respir Crit Care Med 2000;161:S41–5. 6. Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management and Prevention of
ARTICLE IN PRESS 832
7.
8.
9.
10. 11. 12.
13.
14.
15.
E. Gaki et al. Chronic Obstructive Pulmonary Disease. NHLBI/WHO workshop report. Bethesda, National Heart, Lung and Blood Institute, April 2001; Update of the Management Sections, GOLD website /http:\\www.goldcopd.comS. Date updated: July 2005. Global strategy for asthma management and prevention. National Institutes of Health. National Heart, Lung and Blood Institute. NIH Publication No. 02-3659, 2005 updated. Zervas E, Loukides S, Papatheodorou G, Psathakis K, Tsintiris K, Panagou P, et al. Magnesium levels in plasma and in erythrocytes before and after histamine challenge. Eur Respir J 2000;16:621–5. American Thoracic Society. Guidelines for metacholine and exercise challenge testing. Am J Respir Crit Care Med 2000;161:309–29. Standardization of Spirometry 1994. Update, American Thoracic Society. Am J Respir Crit Care Med 1995;152:1107–36. Pizzichini E, Pizzichini MM, Leigh R, Djukanovic R, Sterk PJ. Safety of sputum induction. Eur Respir J Suppl 2002;37:9s–18s. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–10. Vachier I, Kumlin M, Dahlen SE, Bousquet J, Godard P, Chanez P. High levels of urinary leukotriene E4 excretion in steroid treated patients with severe asthma. Respir Med 2000;97:1225–9. Green SA, Malice MP, Tanaka W, Tozzi CA, Reiss TF. Increase in urinary leukotriene LTE4 levels in acute asthma: correlation with airflow limitation. Thorax 2004;59:100–4. Shindo K, Hirai Y, Fukumura M, Koide K. Plasma levels of leukotriene E4 during clinical course of chronic obstructive pulmonary disease. Prostaglandins Leukot Essent Fatty Acids 1997;56:213–7.
16. Lee E, Robertson T, Smith J, Kilfeather S. Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals. Am J Respir Crit Care Med 2000;161:1881–6. 17. Rubinstein I, Kumar B, Schriever C. Long-term montelukast therapy in moderate to severe COPD—a preliminary observation. Respir Med 2004;98:134–8. 18. Yuksel H, Ozbilgin K, Coskun S, Tuglu I. Protective effect of leukotriene receptor antagonist Montelukast on smoking-induced lung injury in wistar rats. Acta Med Okayama 2003;57: 13–9. 19. Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S. Prostaglandin E2 in the expired breath condensate of patients with asthma. Eur Respir J 2003;22:743–7. 20. Tomlinson JE, McMahon AD, Chaudhuri R, Thompson JM, Wood SF, Thomson NC. Efficacy of low and high dose inhaled corticosteroid in smokers versus non-smokers with mild asthma. Thorax 2005;60:282–7. 21. Pavord ID, Ward R, Woltmann G, Wardlaw AJ, Sheller JR, Dworski R. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med 1999;160:1905–9. 22. Fauler JC, Frolich JC. Cigarette smoking stimulates cysteinyl leukotriene production in man. Eur J Clin Invest 1997;27: 43–7. 23. Montuschi P, Kharitonov SA, Ciabattoni G, Barnes PJ. Exhaled leukotrienes and prostaglandins in COPD. Thorax 2003;58: 585–8. 24. Kumlin M, Stensvad F, Larsson L, Dahlen B, Dahlen SE. Validation and application of a new simple strategy for measurements of urinary leukotriene E4 in humans. Clin Exp Allergy 1995;25: 467–79.
Leukotriene E4 in urine in patients with asthma and COPD—The effect of smoking habit E. Gakia, G. Papatheodoroub, E. Ischakia, V. Grammenoua, I. Papaa, S. Loukidesc, a
Pneumonology Department, Athens Veterans Hospital, Athens, Greece Clinical Research Unit, Athens Army General Hospital, Athens, Greece c University of Athens Medical School, Smolika 2, Voula, Athens 16673, Greece b
Received 9 April 2006; accepted 28 June 2006
KEYWORDS Asthma; Chronic obstructive pulmonary disease; Leukotriene E4; Smoking; Eosinophils
Summary Leukotriene E4 (LTE4) is implicated in asthma pathophysiology and possibly in chronic obstructive pulmonary disease (COPD) as one of the causes of persistent bronchoconstriction and mucus hypersecretion. Cigarette smoking stimulates cysteinyl leukotrienes (CysLTs) production. We investigated whether LTE4 is equally increased in asthma and COPD and whether smoking significantly affects LTE4 levels. Secondary outcomes involved correlations with inflammatory and functional parameters. We studied 40 patients with COPD [20 smokers], 40 asthmatics [20 smokers] and 30 healthy subjects [15 smokers]. Spirometry (FEV1% pred., FEV1/FVC) was performed, urine was collected for measurement of LTE4 and creatinine, induced sputum was collected for differential cell counts and serum for ECP. LTE4/creatinine levels (pg/mg) [mean (SD)] were increased in asthmatic patients compared to COPD and controls, [125.6(54.5) vs. 54.5(19) vs. 55.9(18.9) pg/mg, respectively, Po0.0001 for asthma]. Smoking significantly affects LTE4 levels only in asthmatic patients [164 (48) vs. 87 (26.3), Po0.0001 for smokers]. The only significant correlation was between eosinophils in induced sputum and LTE4/creatinine levels in asthmatics. In conclusion, patients with asthma presented higher LTE4 values compared to normals and patients with COPD. Smoking significantly affects LTE4 values only in asthmatics indicating a different underlying CysLTs inflammatory process in this condition. & 2006 Elsevier Ltd. All rights reserved.
Introduction Corresponding author. Tel.: +30 210 8954603;
fax: +30 210 7770423. E-mail address: [email protected] (S. Loukides). 0954-6111/$ - see front matter & 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.rmed.2006.06.031
Cysteinyl leukotrienes (CysLTs) are a group of inflammatory mediators, which has received interest in the recent years. CysLTs (leukotrienes C4, D4 and E4) are central mediators in
ARTICLE IN PRESS Leukotriene E4 in urine in patients with asthma and COPD asthma pathophysiology. Several inflammatory cells have the biosynthetic capacity to produce CysLTs and Leukotriene E4 (LTE4) is a stable end product of CysLTs metabolism in the human lung. Urine LTE4 is a reliable marker of endogenous CysLTs formation and measurement of LTE4 offers a possibility to monitor changes in the rate of CysLTs production.1 Cigarette smoking stimulates CysLTs production in animal models but also in humans.2 The role of CysLTs in chronic obstructive pulmonary disease (COPD) remains uncertain despite previous but limited evidence which indicates increased production in different fluids in this disorder.3,4 However, there is evidence that CysLTs increase microvascular permeability, evoke mucus hypersecretion and are potent smooth muscle constriction agents,5 features that are present in the airways of COPD patients, and this suggests that they might play a role in this disorder. We tested the hypothesis that LTE4 values are equally increased in both asthma and COPD patients, and smoking habit significantly affects the above measurements. Therefore, we assessed the levels of LTE4 in the urine of patients with COPD and asthma, as well as in healthy controls, in an attempt to clarify the in vivo role of this mediator. Smoking as a possible factor that might affect LTE4 values was also tested after subdividing the study groups according to their smoking habit. Additionally, we investigated whether airway or systemic eosinophilic inflammation is implicated in the pathophysiology of this mediator in the two diseases, by assessing sputum differential cell counts and serum eosinophilic cationic protein (ECP) levels. Finally, we examined whether the functional status, as assessed by forced expiratory volume in 1 s (FEV1%) predicted and the FEV1/ FVC (FVC—forced vital capacity) ratio, is implicated in the measurements of LTE4.
Materials and methods Subjects Subjects’ characteristics are summarized in Table 1. The diagnosis of COPD was established according to the GOLD Guidelines,6 whereas the GINA Guidelines were used for the diagnosis of asthma.7 All subjects in the three study groups were subdivided according to their smoking habit (for COPD Table 1
827 patients this subdivision referred to current and exsmokers). A total of 145 subjects were initially recruited in order to achieve the final number of patients. Forty COPD patients (20 current smokers, all non-atopic and steroid-naı¨ve) were recruited. All COPD patients were characterized by the absence of eosinophils in induced sputum. All patients were receiving occasionally short acting b2-agonists as relief medication. Ex-smokers with COPD stopped smoking at least 2 years before entering the study. Forty atopic asthmatics, matched for age, BMI and lung function with COPD patients (20 current smokers, 20 never smokers, matched for age, BMI, lung function and smoking habit with the COPD patients who were current smokers; all steroid-naı¨ve), were also studied. All asthmatic patients had positive bronchodilation tests (increase of pre-bronchodilator FEV1412% after administration of salbutamol at dose 400 mcg by meter dose inhaler). They were occasionally receiving short-acting b2-agonists as relief medication. Thirty normal subjects (15 current smokers, 15 never smokers, matched for the smoking habit with the respective asthmatic and COPD smokers), with no history of any disease, served as controls. All were non-atopic, had normal lung function and negative bronchial challenge testing, and none of them was receiving any kind of medication at the time of the study. Normal subjects were younger compared to asthma and COPD patients since it is difficult to find healthy subjects without any comorbidities in the age of 65 years old. All patients were clinically stable, i.e. they had no evidence of acute exacerbation for at least 4 weeks prior to the study. None of our patients was receiving any antiinflammatory treatment (including corticosteroids, longacting b2 agonists, or leukotriene receptor antagonists), theophylline, inhaled or oral mucolytics, or long-term oxygen therapy. Atopy was defined by the high values of total IgE and on the basis of positive skin tests to six common aeroallergens.
Study protocol details Lung function measurements were performed in all subjects one day before the urine and blood collection and sputum induction. In all subjects, the collection of urine and blood
Subject characteristics.
Age (years) BMI (kg/m2) FEV1 (% pred) FEV1/FVC ratio (%) Smoking habit (pack-years) ECP Eosinophils (%) Neutrophils (%)
Controls (n ¼ 30) 9F–21M
Asthma (n ¼ 40) 14F–26M
COPD (n ¼ 40) 12F–28M
48 (8.5) (33–67) 26 (3.5) (20–35) 99 (10.5) (85–123) 99.5 (9) (89–125) n ¼ 15 38 (9) (19–80) 6(2) (3–9) ND ND
63 (14) (31–80) 28.4 (4) (20–40) 71 (16) (42–98) 68 (4) (59–76) n ¼ 20 37.5 (10) (20–55) 14.5(7) (5–35) 7.5 (4) (2–17) 28.5 (6) (22–40)
64 (8) (49–81) 27(4) (21–34) 67 (9) (49–85) 64 (3) (57–70) n ¼ 20 41 (11) (25–63) 6.5(2) (3–10.5) 0 64(7) (45–73)
Data are presented as mean (SD) with ranges in parenthesis. ND ¼ not done, M ¼ male, F ¼ female. Smoking habit refers only to the subgroups of current smokers in each group.
ARTICLE IN PRESS 828
E. Gaki et al.
preceded the sputum induction procedure, in order to avoid any effect of sputum induction procedure on the urine and blood measurements. All subjects were asked not to smoke 2 h before each procedure. In order to ensure the subjects’ compliance to this recommendation, they were asked to stay in our laboratory for 2 h under the supervision of one of the investigators. Sputum induction was not performed in normal subjects. Metacholine test was performed only in normal subjects on a separate day of the protocol. All subjects were asked not to receive short-acting bronchodilators at least 12 h before the collection of samples and the lung function tests. LTE4/creatinine values were compared among patients with COPD, patients with asthma and normal subjects. Similar comparisons were performed in the study subgroups. Correlations between LTE4/creatinine values and study parameters were also performed in all study groups. All subjects were recruited from the outpatient clinics of the Athens Veterans Hospital. This is a general hospital that serves veterans and civilians. The Scientific Committee of the hospital approved the study protocol, and all participants gave written informed consent.
immunoassay (EIA) (Cayman Chemical; Ann Arbor, MI, USA, No. 520411). The detection limit of the assay was 25 pg/mL. The results were normalized to the creatinine concentration determined in the same sample and the LTE4/creatinine ratios were used for subsequent studies. All urine samples were tested for protein, bilirubin, ketone, urobilinogen, glucose and hemoglobin and if any of these substances were presented the sample was excluded from the study. According to manufacturer’s guidelines and in order to check the need of purification, all samples diluted 1:3 with distilled water. LTE4 was measured in diluted and undiluted samples. The two different dilutions of the samples showed differences less than 18% in the final calculated LTE4 concentration, so the samples were measured without purification.
Metacholine challenge
ECP measurement
Bronchial hyperresponsiveness to metacholine was measured and performed by a rapid metacholine inhalation test for the determination of PD20, as previously described for histamine.8 PD20 was determined by linear interpolation on a semi-logarithmic scale. The test was performed according to the instructions established by the American Thoracic Society guidelines.9
The concentrations of ECP in the serum were determined by a specific enzyme immuno-assay (Pharmacia Diagnostics AB; Uppsala, Sweden) in an auto analyzer (Uni CAP 100) with a lower limit of detection of 2.0 mg/L.
Lung function Pulmonary function tests were measured with a dry spirometer (Vica-test, Model VEP2; Mijnhardt; Rotterdam, Holland). FEV1 and FVC were measured according to the American Thoracic Society guidelines.10
Creatinine measurement Creatinine concentration in urine was measured with a kinetic method. The complex formed by creatinine and picric acid (8.8 mmol/L) in an alkaline medium (NaOH 0.4 M) was measured for 1 min at 492 nm.
LTE4/creatinine repeatability measurements Repeatability of LTE4/creatinine measurements was evaluated in 20 subjects [4 controls (2 smokers),8 COPD patients (4 from each subgroup) and 8 asthmatics (4 smokers)]. Repeatability of LTE4/creatinine measurements was tested in samples from the same subjects collected on two consecutive days.
Statistical analysis Sputum induction and processing Sputum was induced as previously described.11 Subjects were asked to blow their noses, rinse their mouths, and swallow the water in the aerosol to minimize contamination with postnasal drip and saliva. An induction procedure using inhalation of an aerosol of hypertonic saline solution (3.5%) generated by a DeVilbiss ultrasonic nebulizer (2696 Somerset PA, USA) was chosen. Induction was performed for periods of 30 s, and 1, 2, 4 and 8 min. The patients were asked to expectorate sputum after the 4 and 8 min period. At least 2 mL of sputum were collected in a sterile container. The person who did the differential cell counts in sputum (G.P.) was not aware of the clinical and functional status of the patients or the results of bronchial challenge testing.
LTE4 measurements LTE4 concentrations were measured in a non-purified urine morning sample with a specific competitive enzyme
Data are expressed as mean (SD), unless otherwise mentioned. Values among three or more groups were evaluated using one-way analysis of variance (ANOVA) with an appropriate post hoc test for multiple comparisons (Bonferroni). For comparisons between two groups, Mann–Whitney U tests were used. Spearman’s correlation coefficient was used to investigate correlations between parameters. Repeatability of LTE4/creatinine measurements was assessed with the Bland and Altman test.12 A P-value o0.05 was considered significant.
Results Subjects characteristics in induced sputum Asthmatic patients were mainly characterized by a predominant eosinophilia in induced sputum whereas COPD patients were characterized by a predominance of neutrophilic inflammation (Table 1). Asthmatic smokers presented
ARTICLE IN PRESS
Ex-smokers (n ¼ 20) 5F–15M
with significantly higher values of % eosinophils in induced sputum compared to non-smokers (Table 2, Po0.003). Asthmatic smokers presented a trend for higher values of % neutrophils in induced sputum compared to non-smokers, that was marginally statistically significant (Table 2, P ¼ 0.048). No significant differences were observed between current and ex-smokers patients with COPD in regard to % neutrophils in induced sputum (Table 2, P ¼ 0.79)
The measurements of LTE4/creatinine on 2 consecutive days presented good repeatability. LTE4/creatinine on days 1 and 2 were 80.96(52) pg/mg and 82.04(52) pg/mg, respectively. The correlation between LTE4/creatinine measurements on two consecutive days was significant (r ¼ 0.94, Po0.0001). The mean difference with limits of agreement was 1.0874.9 (mean72 SD) in the Bland and Altman plot.
Discussion In this prospective cross-sectional study we have found significant elevation of LTE4 levels in the urine of patients
13.5 (5) (5.5–23) 4.5 (2) (2–9) 25 (6.5) (22–35)
65(6) (49–73) 27 (4) (21–33) 66 (8.5) (49–81) 64 (3) (57–70) 41 (11) (25–63) 6 (2) (4–9) 0 66(8) (45–73) 62 (16.5) (31–79) 27.5(4) (20–34.5) 72 (17) (42–98) 69 (4.5) (60–76)
5 (2) (3–9) ND ND
65 (11) (47–80) 29(4) (24–40) 70.5 (15) (53–97) 68(4) (59–72) 37.5 (10) (20–55)) 16 (9) (5–35) 10 (4)** (4–17) 31.5 (6) (28–40) 46 (7) (34–59) 27(3) (21–35) 100(11) (85–123) 102.5 (10) (89–125)
Smokers (n ¼ 20) 7F–13M Smokers (n ¼ 15) 4F–11M
Non-smokers (n ¼ 15) 5F–10M
Asthma
Repeatability of LTE4/creatinine measurements
Table 2
Major correlation data are summarized in Table 3. Briefly, the values of LTE4/creatinine presented a significant correlation with eosinophils in induced sputum in asthmatics (r ¼ 0.83, Po0.0001). This significant correlation was also observed in smoking asthmatics, but was not expressed in asthmatic non-smokers (Table 3, Figs. 3A and B). No correlation with ECP levels was found. No significant correlation was observed between the levels of LTE4/ creatinine and any of the parameters tested in COPD patients. No significant correlation was observed between LTE4 and the degree of lung function impairment or the smoking habit in pack-years in all study groups. Neutrophils in COPD patients did not present any significant correlations with LTE4/creatinine (r ¼ 0.02, P ¼ 0.77).
Subject characteristics according to smoking habit.
Correlations
Controls
The levels of LTE4/creatinine (pg/mg) were increased in asthmatic smokers compared to non-smokers [164 (48) vs. 87 (26.3) pg/mg, Po0.0001, Fig. 2]. No significant difference was observed between smokers and non-smokers in the COPD and control groups [58.3 (23) vs. 50.5(13) pg/mg, P ¼ 0.4, 56(20) vs. 56 (18), P ¼ 0.83, respectively, Fig. 2]. However, a trend for higher values in COPD smokers compared to ex-smokers was evident.
49.5 (9.5) (33–67) 26(4) (20–35) 98 (10.5) (87–123) 96.5 (6) (89–106) 38 (9) (19–80) 6.7 (1.5) (4.5–9) ND ND
Non smokers (n ¼ 20) 7F–13M
Effect of smoking
Age (years) BMI (kg/ m2) FEV1 (% pred) FEV1/FVC ratio (%) Smoking habit (pack–years) ECP Eosinophils (% ) Neutrophils (%)
COPD
The levels of LTE4/creatinine (pg/mg) were increased in asthmatic subjects compared to COPD and controls [125.6(54.5) vs. 54.5(19) vs. 55.9(18.9) pg/mg, respectively, Po0.0001 for asthma, Fig. 1]. No statistically significant difference was found between COPD patients and normal subjects [54.5(19) vs. 55.9(18.9) pg/mg, P ¼ 0.3, Fig. 1].
Smokers (n ¼ 20) 7F–13M
LTE4/creatinine levels in major study groups
Data are presented as mean (SD) with ranges in parenthesis. ND ¼ not done, M ¼ male, F ¼ female, *ex-smoking history, **Significant higher compared to non-smoking asthmatics, P ¼ 0.003, Non-smokers ¼ never smokers.
829
62 (7.5) (51–81) 26.5(4) (22–34) 68 (8) (52–85) 65(4) (58–70) 42 (9)* (32–60) 6.5(2) (3–10.5) 0 62 (5.5) (48–70)
Leukotriene E4 in urine in patients with asthma and COPD
ARTICLE IN PRESS 830
E. Gaki et al. p<0.0001
LTE4 / creatinine pg/mg
300 250 200 150 100 50 0 Normals
Asthma
COPD
Figure 1 LTE4/creatinine concentration in control subjects [all n ¼ 30 ’], patients with asthma [all n ¼ 40 m, and patients with COPD [all n ¼ 40 ., Each symbol represents one individual. Horizontal bars represent mean values. See text for details.
LTE4/creatinine pg/mg
300
p<0.0001
250 200
p=0.83 p=0.4
150 100 50 0 NS
S
Normals
NS
S
Asthma
ES
S
COPD
Figure 2 LTE4/creatinine concentration in non-smoking control subjects [n ¼ 15 &], smoking control subjects [n ¼ 15 ’], non-smoking patients with asthma [n ¼ 20 ,], smoking patients with asthma [n ¼ 20m], ex-smokers with COPD [n ¼ 20 J] and smokers with COPD [n ¼ 20 K]. Each symbol represents one individual. Horizontal bars represent mean values. S ¼ smoking, NS ¼ non-smoking, ES ¼ Ex-smoking. See text for details.
Table 3
with asthma compared to matched COPD patients and healthy controls. Smoking habit significantly affects LTE4 levels only in asthmatic subjects. The values of LTE4 in urine present significant correlations with eosinophils in induced sputum only in asthmatics; interestingly, the absence of smoking habit seems to affect negatively the above significance. The measurements of LTE4/creatinine on 2 consecutive days are repeatable. CysLTs are synthesized from arachidonic acid through a 5-lipoxygenase pathway and are mainly generated by many inflammatory cells, particularly eosinophils, mast cells and macrophages. The implication of LTE4 in asthmatic inflammation is well established, and the increased levels of LTE4 in the urine of asthmatic subjects reported in this study are comparable to those published by previous investigators.13,14 However, this is the first study to our knowledge that evaluates the levels of LTE4 in urine of patients with COPD. Two previous studies have reported increased CysLTs levels in patients with COPD; however, in the first study the mediator that was used (LTC4) is not considered as a stable end product in CysLT production,2 and in the second the biological fluids studied did not represent sources which would definitely express airway inflammation.15 Interestingly, the levels measured were not significantly higher compared to normal subjects, indicating that the 5-lipoxygenase pathway of arachidonic acid metabolism may not be activated in COPD patients. The reason for this observation might be attributed to the capability and/or the activation state of the inflammatory cells which participate in the CysLTs production. This is partially supported by the findings of our study that show a positive correlation between sputum eosinophils and LTE4 values in asthma and the absence of any correlation between induced sputum cells and LTE4 values in COPD. However, this positive correlation might also express the CysLTs-induced prolonged survival of eosinophils in the airways that contributes to the maintenance of the inflammatory process in asthma.16 The absence of similar correlation with ECP might be related to the low sensitivity of this mediator for the assessment of eosinophilic inflammation. A previous observation, where the use of a CysLT1 receptor antagonist led to improvement of some COPD patients with less fixed airflow obstruction was not tested in this study, since the methodology used did not include a proper
Correlations of LTE4 in COPD and asthmatic patients as well as in asthmatic smokers and non-smokers.
LTE4 Asthma
FEV1 (% predicted) FEV1/FVC ratio Eosinophils (%) Smoking (py) ECP (mg/L) Neutrophils
COPD
Asthma smokers
Asthma non-smokers
r
P-value
r
P-value
r
P-value
r
P-value
0.02 014 0.83 ND 0.10 0.03
0.80 0.35 o0.0001 ND 0.50 0.86
0.04 0.21 ND ND 0.002 0.02
0.74 0.10 ND ND 0.83 0.77
0.07 0.1 0.77 0.24 0.21 0.04
0.74 0.63 o0.0001 0.25 0.73 0.81
0.10 0.07 0.31 ND 0.08 0.06
0.6 0.77 0.10 ND 0.71 0.88
Bold letters indicate significant correlations. ND ¼ not done.
ARTICLE IN PRESS Leukotriene E4 in urine in patients with asthma and COPD
r=0.77 p<0.0001
16 14 12 10 8 6
10 % eosinophilsin induced sputum in asthmatic non smokers
% eosinophils in induced sputum in asthmatics smokers
18
831
8 6 4 2
4 100 (A)
r=0.31 p=0.1
125 150 175 200 225 250 LTE4/creatinine pg/mg in asthmatics smokers
40 (B)
60 80 100 120 140 160 LTE4/creatinine pg/mg in asthmatics non smokers
Figure 3 Correlations between the concentration of LTE4/creatinine and eosinophils (A) in asthmatic smokers (r ¼ 0.77, Po0.0001) and (B) in asthmatic non-smokers (r ¼ 0.35, P ¼ 0.1).
placebo-controlled design; thus, any conclusions arising were not strong enough to confirm a potential role of CysLT1 receptor antagonists in COPD.17 Smoking affects significantly LTE4 levels in asthma but not in COPD and normal subjects. The positive effect of smoking in CysLTs production in asthma, irrespective of the degree of smoking habit, might be related to the number and/or the activation state of cells responsible for producing CysLTs. Cells like alveolar macrophages and mast cells, which are activated in asthma and also increased by smoking, might stimulate the production of CysLTs via the preexisting activated 5-lypoxygenase pathway. This theory is supported by previous observations where in vivo data showed the protective effect of a leukotriene receptor antagonist on cigarette smoking-induced lung injury.18 Similar findings regarding the concentration of the eicosanoid PGE2 in smoking asthmatics19 indicated that eicosanoid metabolism in asthmatics is significantly influenced by smoking. Data from the present study showing absence of significant correlation between eosinophils in induced sputum and the levels of LTE4 in the urine of non-smoking asthmatics may represent further evidence in this direction. However a possible limitation of our study by combining the above observations might be that they just represent the prolonged survival of eosinophils that is induced by the increased CysLTs or the eosinophil-induced CysLTs production. The above observation about the effect of smoking on asthmatics might have practical implications, since it might partially explain the difference in the response to inhaled steroid treatment in smoking asthmatics, once the CysLTsrelated pathophysiology is not sensitive to the treatment with steroids.20,21 Interestingly, the above observations were not present in normal subjects and COPD patients. This finding does not seem reasonable, but it could be attributed to the previously inactivated 5-lipoxygenase pathway. In normal subjects it might also be explained by previous observations where the increase in LTE4 was attributed to acute effects rather than to indirect effects resulting from chronic smoking exposure, since 5-lipoxygenase is not in a proper state to be activated and to produce CysLTs.2 However, some previous data supports that smoking in a dose-dependent manner significantly affects LTE4 values in normal subjects.22
The repeatability data presented in this study are similar to data reported for COPD using another biological fluid,23 indicating that, in stable patients with chronic inflammatory airway diseases and persistent inflammation, the measurement of mediators involved in the inflammatory process may be repeatable. This study presents one possible limitation, regarding the measurement of LTE4 in urine, which was performed by an EIA without purification. However, LTE4 has been shown to be stable in urine samples stored at 20 1C for months without the addition of preservatives. Additionally, LTE4 levels in crude urine samples assessed by EIA have been shown to present significant correlation with LTE4 measurements performed after purification on solid-phase extraction followed by separation on reversed-phase highperformance liquid chromatography24 and finally a specific procedure assessing interference in diluted and undiluted samples as suggested by the manufacturer was performed. In summary, we report that patients with asthma present higher urine LTE4 values compared to patients with COPD and to healthy controls. Smoking in asthmatics seems to be a critical parameter that might influence the above values. Eosinophils as a cellular source or as a result of prolonged survival are probably the key inflammatory cells in CysLTsrelated inflammatory process in smoking asthmatics.
References 1. Wennergren G. Inflammatory mediators in blood and urine. Paediatr Respir Rev 2000;1:259–65. 2. Saareks V, Riutta A, Alanko J, Ylitalo P, Parviainen M, Mucha I, et al. Clinical pharmacology of eicosanoids nicotine induced changes in man. J Physiol Pharmacol 2000;51:631–42. 3. Mitsunobu F, Mifune T, Hosaki Y, Ashida K, Tsugeno H, Okamoto M, et al. Enhanced production of leukotrienes by peripheral leukocytes and specific igE antibodies in patients with COPD. J Allergy Clin Immunol 2001;107:492–8. 4. Piperno D, Pacheco Y, Hosni R, Moliere P, Gharib C, Lagarde M, et al. Increased plasma levels of atrial natriuretic factor, renin activity and leukotriene C4 in chronic obstructive pulmonary disease. Chest 1993;104:454–9. 5. Dahlen SE. Pharmacological characterization of leukotrienes receptors. Am J Respir Crit Care Med 2000;161:S41–5. 6. Global Initiative for Chronic Obstructive Lung Disease. Global Strategy for the Diagnosis, Management and Prevention of
ARTICLE IN PRESS 832
7.
8.
9.
10. 11. 12.
13.
14.
15.
E. Gaki et al. Chronic Obstructive Pulmonary Disease. NHLBI/WHO workshop report. Bethesda, National Heart, Lung and Blood Institute, April 2001; Update of the Management Sections, GOLD website /http:\\www.goldcopd.comS. Date updated: July 2005. Global strategy for asthma management and prevention. National Institutes of Health. National Heart, Lung and Blood Institute. NIH Publication No. 02-3659, 2005 updated. Zervas E, Loukides S, Papatheodorou G, Psathakis K, Tsintiris K, Panagou P, et al. Magnesium levels in plasma and in erythrocytes before and after histamine challenge. Eur Respir J 2000;16:621–5. American Thoracic Society. Guidelines for metacholine and exercise challenge testing. Am J Respir Crit Care Med 2000;161:309–29. Standardization of Spirometry 1994. Update, American Thoracic Society. Am J Respir Crit Care Med 1995;152:1107–36. Pizzichini E, Pizzichini MM, Leigh R, Djukanovic R, Sterk PJ. Safety of sputum induction. Eur Respir J Suppl 2002;37:9s–18s. Bland JM, Altman DG. Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1986;1:307–10. Vachier I, Kumlin M, Dahlen SE, Bousquet J, Godard P, Chanez P. High levels of urinary leukotriene E4 excretion in steroid treated patients with severe asthma. Respir Med 2000;97:1225–9. Green SA, Malice MP, Tanaka W, Tozzi CA, Reiss TF. Increase in urinary leukotriene LTE4 levels in acute asthma: correlation with airflow limitation. Thorax 2004;59:100–4. Shindo K, Hirai Y, Fukumura M, Koide K. Plasma levels of leukotriene E4 during clinical course of chronic obstructive pulmonary disease. Prostaglandins Leukot Essent Fatty Acids 1997;56:213–7.
16. Lee E, Robertson T, Smith J, Kilfeather S. Leukotriene receptor antagonists and synthesis inhibitors reverse survival in eosinophils of asthmatic individuals. Am J Respir Crit Care Med 2000;161:1881–6. 17. Rubinstein I, Kumar B, Schriever C. Long-term montelukast therapy in moderate to severe COPD—a preliminary observation. Respir Med 2004;98:134–8. 18. Yuksel H, Ozbilgin K, Coskun S, Tuglu I. Protective effect of leukotriene receptor antagonist Montelukast on smoking-induced lung injury in wistar rats. Acta Med Okayama 2003;57: 13–9. 19. Kostikas K, Papatheodorou G, Psathakis K, Panagou P, Loukides S. Prostaglandin E2 in the expired breath condensate of patients with asthma. Eur Respir J 2003;22:743–7. 20. Tomlinson JE, McMahon AD, Chaudhuri R, Thompson JM, Wood SF, Thomson NC. Efficacy of low and high dose inhaled corticosteroid in smokers versus non-smokers with mild asthma. Thorax 2005;60:282–7. 21. Pavord ID, Ward R, Woltmann G, Wardlaw AJ, Sheller JR, Dworski R. Induced sputum eicosanoid concentrations in asthma. Am J Respir Crit Care Med 1999;160:1905–9. 22. Fauler JC, Frolich JC. Cigarette smoking stimulates cysteinyl leukotriene production in man. Eur J Clin Invest 1997;27: 43–7. 23. Montuschi P, Kharitonov SA, Ciabattoni G, Barnes PJ. Exhaled leukotrienes and prostaglandins in COPD. Thorax 2003;58: 585–8. 24. Kumlin M, Stensvad F, Larsson L, Dahlen B, Dahlen SE. Validation and application of a new simple strategy for measurements of urinary leukotriene E4 in humans. Clin Exp Allergy 1995;25: 467–79.