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Provided by Elsevier - Publisher Connector Egyptian Journal of Chest Diseases and Tuberculosis (2016) 65, 23–27
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ORIGINAL ARTICLE Role of neopterin among COPD patients
Ashraf Abdel Halim a,*, Zeinab Adawy b, Manal Sayed c
a Chest Department, Faculty of Medicine, Zagazig University, Egypt b Chest Department, Faculty of Medicine, Al-azhar University, Egypt c Clinical Pathology Department, Egyptian Central Laboratories, Egypt
Received 2 November 2015; accepted 19 November 2015 Available online 10 December 2015
KEYWORDS Abstract Chronic Obstructive Pulmonary Disease (COPD) is a major public health problem. It is the Neopterin; fourth leading cause of chronic morbidity and mortality worldwide. COPD is characterized by a specific COPD; pattern of inflammation involving many cells and mediators. Neopterin serves as a marker of systemic Oxidative stress immune activation, which is of importance in the pathogenesis and progression of various diseases. Aim of the work: This study was conducted to elucidate the role of neopterin in the pathogenesis of COPD. Methods: 55 COPD patients and 20 healthy control subjects of similar age and sex were included in the study. Neopterin levels in the serum and sputum samples were measured in all subjects. Results: The mean values of neopterin in the serum (20.34 ± 4.70 nmol/l) and sputum (32.07 ± 8.14 nmol/l) samples of COPD patients were significantly higher than the mean values of neopterin in the serum and sputum samples of control subjects (6.27 ± 3.35 nmol/l and 4.13 ± 2.25 nmol/l respectively) (p < 0.001). Also, COPD patients with exacerbation had significantly higher (p < 0.001) serum and sputum neopterin levels (23.47 ± 2.56 nmol/l and 39.94 ± 5.47 nmol/l respec- tively) in comparison with stable COPD patients (18.69 ± 4.76 nmol/l and 27.91 ± 5.95 nmol/l respectively). There was statistically significant difference (p value < 0.05) between different COPD severity groups in the mean values of serum and sputum neopterin (nmol/l). There was statistically significant difference (p value < 0.05) in the mean values of serum and sputum neop- terin between different COPD patients when they are classified according to the smoking state. Current smokers have higher mean values of serum (22.40 ± 3.04 nmol/l) and sputum (39.60 ± 7.56 nmol/l) neopterin than ex smokers (20.33 ± 4.67 nmol/l and 29.84 ± 6.33 nmol/l respectively) and nonsmok- ers (16.00 ± 5.3 nmol/l and 26.42 ± 6.60 nmol/l respectively). Conclusions: This study found increased neopterin serum and sputum levels among COPD patients and suggests a role for neopterin in the pathogenesis of COPD. We recommend larger studies to sup- port our results. Ó 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).
* Corresponding author. Tel.: +20 1100214851. E-mail address: [email protected] (A.A. Halim). Peer review under responsibility of The Egyptian Society of Chest Diseases and Tuberculosis. http://dx.doi.org/10.1016/j.ejcdt.2015.11.010 0422-7638 Ó 2015 The Authors. Production and hosting by Elsevier B.V. on behalf of The Egyptian Society of Chest Diseases and Tuberculosis. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 24 A.A. Halim et al.
Introduction Stable Chronic Obstructive Pulmonary Disease
Chronic Obstructive Pulmonary Disease (COPD) is a major Stable COPD is defined by the absence of any exacerbation for public health problem. It is the fourth leading cause of 3 months preceding the study [8]. chronic morbidity and mortality in the United States, and is projected to rank fifth in 2020 in burden of disease caused Acute exacerbation of COPD (AECOPD) worldwide, according to a study published by the World Bank/World Health Organization [1]. COPD is characterized The American Thoracic Society (ATS) and European Respira- by a specific pattern of inflammation involving neutrophils, tory Society (ERS) define an exacerbation as an acute change macrophages, and lymphocytes. These cells release many in a patient’s baseline dyspnea, cough, or sputum that is inflammatory mediators and interact with structural cells in beyond normal variability, and that is sufficient to warrant a the airways and lung parenchyma [2]. A wide variety of change in therapy [9]. inflammatory mediators that have been shown to be increased in COPD patients attract inflammatory cells from Methods the circulation (chemotactic factors), amplify the inflamma- tory process (proinflammatory cytokines), and induce struc- All persons included in this study were subjected to the tural changes [3]. following: Neopterin is a catabolic product of guanosine triphos- phate (GTP), a purine nucleotide. Neopterin belongs to the 1. Full history taking. chemical group known as pteridines. It is synthesized by acti- 2. Thorough medical examination. vated monocytes, macrophages, dendritic cells, and endothe- 3. Plain chest radiography (posterio-anterior and lateral). lial cells upon stimulation with the cytokine interferon- 4. Pulmonary function testing. gamma and to a lesser extent by interferon alpha and beta 5. Complete blood picture (CBC) and differential. with its release being enhanced by tumor necrosis factor 6. CRP. [4,5]. Neopterin serves as a marker of systemic immune 7. Measurement of neopterin levels in the serum and sputum activation. Measurement of neopterin concentrations in body samples of COPD patients and control subjects. fluids like blood serum, cerebro-spinal fluid or urine provides information about cellular immune activation, which is of Blood samples for laboratory assessments were obtained importance in the pathogenesis and progression of various from all subjects included in this study. Sera were separated diseases, e.g., in viral infections, in autoimmune or inflamma- by centrifugation at 1500 g for 10 min and were frozen at tory diseases, rejection episodes following allograft transplan- À70 °C and protected from light. Sputum samples were also tation, autoimmune-logical diseases and in several malignant collected from all included subjects. All COPD patients gave diseases [6,7]. their sputum samples spontaneously, but control subjects gave their samples after the induced sputum technique [10]. Sputum Aim of the work samples were kept in a cold place (temp. 4 °C) and processed within two hours. They were solubilized with DTT (dithiothre- This study was conducted to elucidate the role of neopterin in itol) followed by Dulbecco’s phosphate buffer saline (D-PBS). the pathogenesis of COPD. Then the mixture was filtered and centrifuged. The super- natant is aspirated and frozen at À70 °C for neopterin Subjects and methods analysis. Neopterin concentrations were determined in the serum and sputum samples by the ELISA method using This study included 55 patients with COPD (45 men, 10 commercially available kit manufactured by IBL, Hamburg women) with a mean age of 56.49 ± 8.52 years (ranging 42–78) (Germany) [11]. that were selected from patients who visited our university hospitals and 20 healthy control subjects (15 men, 5 women, mean age 54.37 ± 7.33 years, range 39–74 years) (Table 1). Results Patients and control subjects were matched for age and gender. The COPD patient group included 36 stable COPD patients The mean values of neopterin in the serum (20.34 ± and 19 COPD patients with exacerbation. 4.70 nmol/l) and sputum (32.07 ± 8.14 nmol/l) samples of The diagnosis of COPD was established by clinical symp- COPD patients were significantly higher than the mean values toms, physical examination, chest radiography and pulmonary of neopterin in the serum and sputum samples of control sub- function tests according to guidelines of GOLD (2014) [1]. jects (6.27 ± 3.35 nmol/l and 4.13 ± 2.25 nmol/l respectively) The COPD subjects were classified into (stages I–IV) on the (p < 0.001) (Table 2 and Fig. 1). Also, COPD patients with basis of their post-bronchodilator forced expiratory volume in exacerbation had significantly higher (p < 0.001) serum and one second (FEV1)/forced vital capacity (FVC) and FEV1 % sputum neopterin levels (23.47 ± 2.56 nmol/l and 39.94 predicted. FEV1/FVC was <70% and their FEV1 fell into set ± 5.47 nmol/l respectively) in comparison with stable COPD bands (stage 1: FEV1 P 80%; stage 2: 50% P FEV1 < 80%; patients (18.69 ± 4.76 nmol/l and 27.91 ± 5.95 nmol/l respec- stage 3: 30% P FEV1 < 50%; stage 4: FEV1 < 30%). tively) (Table 3 and Fig. 2). Role of neopterin among COPD patients 25
Table 1 Characteristics of COPD patients and control Table 3 Mean values of serum and sputum neopterin among groups. stable COPD patients and COPD patients with exacerbation. Group Patients Control Group Serum neopterin Sputum neopterin p value mean values ± SD mean values ± SD No. 55 20 Male/female 45/10 15/5 (nmol/l) (nmol/l) Age yrs 56.49 ± 8.52 54.37 ± 7.33 Stable 18.69 ± 4.76 27.91 ± 5.95 <0.001 FEV1 % pred 51.88 ± 17.52 93.66 ± 5.42 COPD No. FEV1/FVC % 60.7 ± 5.33 90.75 ± 4.1 =36 PO2 62.54 ± 15.25 95.88 ± 2.70 Exacerbated 23.47 ± 2.56 39.94 ± 5.47 PCO2 41.10 ± 7.55 39.40 ± 2.62 COPD No. Smoking =19 Current 15 0 Ex smoker 33 0 Non smoker 7 20 Severity I (FEV1 P80% of predicted) 9 (16.36%) – Mean neopterin levels (nmol/l) II (FEV1 P50% and <80%) 16 (29.10%) – 45 III (FEV1 P30% and <50%) 20 (36.36%) – 40 IV (FEV1 <30% of predicted) 10 (18.18%) – 35 30 25 20 Table 2 Mean values of serum and sputum neopterin among 15 COPD patients and control groups. 10 5 Group Serum neopterin Sputum neopterin p value mean values ± SD mean values ± SD 0 Serum neopterin Sputum neopterin (nmol/l) (nmol/l) Stable COPD Exacerbated COPD COPD 20.34 ± 4.70 32.07 ± 8.14 <0.001 patients Figure 2 Mean values of serum and sputum neopterin among No. = 55 stable COPD patients and COPD patients with exacerbation. Control 6.27 ± 3.35 4.13 ± 2.25 subjects No. = 20
Table 4 Mean values of serum and sputum neopterin (nmol/l) among different COPD severity groups. Severity Serum neopterin Sputum neopterin Neopterin mean levels(nmol/l) (nmol/l) (mean ± SD) (nmol/l) (mean ± SD) 35 Mild 14.50 ± 2.58 24.16 ± 3.06 (No. = 6) 30 Moderate 20.36 ± 5.34 29.42 ± 7.12 25 (No. = 19) 20 Severe 20.92 ± 3.60 34.48 ± 8.36 (No. = 25) 15 Very severe 24.40 ± 3.20 39.60 ± 0.89 10 (No. = 5)
5 ANOVA F 5.57 6.02 p value <0.01 <0.01 0 Serum neopterin Sputum neopterin
COPD pa ents Control subjects
Figure 1 Mean values of serum and sputum neopterin among serum (r = 0.482) and sputum (r = 0.611) neopterin levels COPD patients and control groups. (Figs. 3 and 4). Also, there is positive correlation between the serum and sputum neopterin levels (r = 0.679). There was statistically significant difference (p value There was a statistically significant difference (p value < <0.05) in the mean values of serum and sputum neopterin 0.05) between different COPD severity groups in the mean val- between different COPD patients when they are classified ues of serum and sputum neopterin (nmol/l) (Table 4). according to the smoking state. Current smokers have higher The serum and sputum neopterin levels increased with mean values of serum (22.40 ± 3.04 nmol/l) and sputum increased severity of COPD. There was a significant positive (39.60 ± 7.56 nmol/l) neopterin than ex smokers (20.33 ± correlation (p value < 0.001) between COPD severity and 4.67 nmol/l and 29.84 ± 6.33 nmol/l respectively) and 26 A.A. Halim et al.
30 [12] evaluated circulating levels of interferon-gamma (IFN- gamma), soluble interleukin-2 receptor (sIL-2R), neopterin, 25 and soluble intercellular adhesion molecule-1 (sICAM-1) in 20 35 clinically stable patients with COPD and in 22 age- matched healthy controls. They found that circulating levels 15 of sIL-2R (1.52+/À 1.25 vs. 0.97+/À 0.48 ng/ml; p < 0.05), À À 10 neopterin (7.23+/ 4.24 vs. 4.95+/ 1.52 nmol/l; p < 0.05), and sICAM-1 (665+/À 302 vs. 328+/À 164 ng/ml; 5 p < 0.0001), but not IFN-gamma (7.55+/À 4.72 vs. 6.65+/À 1.13 pg/ml; p = NS) were significantly higher in 0 Mild Moderate Severe very severe patients with COPD than those in the controls. Warwick et al. [13] detected that, among COPD patients, levels of the Figure 3 Mean values of serum neopterin (nmol/l) among novel markers neopterin and IP-10 were significantly increased different COPD severity groups. in induced sputum supernatant (pooled groups pre and post exacerbation: IP-10: 188.6 ± 102.1 vs. 5.40 ± 1.28 pg/ml, p = 0.006; neopterin: 15.81 ± 2.50 vs. 5.38 ± 0.45 nmol/L, 45 p < 0.0001), as was TNF-a (137.8 ± 49.64 vs. 71.56 ± 40 45.03 pg/ml, p = 0.018). 35 Our study revealed a statistically significant difference 30 (p value < 0.05) between different COPD severity groups in 25 the mean values of serum and sputum neopterin (nmol/l). 20 The serum and sputum neopterin levels increased with 15 increased severity of COPD. There was a significant positive 10 correlation (p value < 0.001) between COPD severity and 5 serum (r = 0.482) and sputum (r = 0.611) neopterin levels. 0 Also, there is a positive correlation between the serum and Mild Moderate Severe very severe sputum neopterin levels (r = 0. 679). Our findings are in agreement with that of others. Garrod et al. [14] found that Figure 4 Mean values of sputum neopterin (nmol/l) among measures of systemic inflammation (neopterin, TNF-a, IL6, different COPD severity groups. and in particular CRP) are correlated with COPD severity in primary care. We detected a statistically significant difference Table 5 Mean values of serum and sputum neopterin (nmol/l) (p value < 0.05) in the mean values of serum and sputum among COPD patients regarding smoking status. neopterin between different COPD patients when they are classified according to the smoking state. Current smokers Group Serum neopterin Sputum neopterin p value have higher mean values of serum (22.40 ± 3.04 nmol/l) and (mean ± SD) (mean ± SD) sputum (39.60 ± 7.56 nmol/l) neopterin than ex smokers Smokers 22.40 ± 3.04 39.60 ± 7.56 p < 0.05 (20.33 ± 4.67 nmol/l and 29.84 ± 6.33 nmol/l respectively) (No. = 15) and nonsmokers (16.00 ± 5.3 nmol/l and 26.42 ± 6.60 nmol/l Ex smokers 20.33 ± 4.67 29.84 ± 6.33 respectively). Other researchers revealed similar results. (No. = 33) Non smokers 16.00 ± 5.35 26.42 ± 6.60 Djordjevic et al. [15] found a strong correlation between (No. = 7) neopterin and both smokers (p < 0.01). Oxidative stress induced by reactive oxygen and nitrogen species (ROS and RNS) plays a central role in the pathophys- iolgy of COPD [16]. Previous data indicate a possible partici- pation of neopterin derivates within the cytotoxic mechanism nonsmokers (16.00 ± 5.3 nmol/l and 26.42 ± 6.60 nmol/l of white blood cells [17]. On the one hand, a strong correlation respectively) (Table 5). between detection of neopterin and ability of monocytes/ macrophages/neutrophils to set free reactive oxygen species Discussion [18] was found. Thus, neopterin determination can be consid- ered as an indirect marker for the amount of immunologically In this study, the mean values of neopterin in the serum (20.34 induced oxidative stress. On the other hand, neopterin ± 4.70 nmol/l) and sputum (32.07 ± 8.14 nmol/l) samples of derivates are able to influence the effects of reactive oxygen, COPD patients were significantly higher than the mean values nitrogen and chlorine species. It was demonstrated that neop- of neopterin in the serum and sputum samples of control sub- terin is a potent enhancer of various reactive substances, such jects (6.27 ± 3.35 nmol/l and 4.13 ± 2.25 nmol/l respectively) as H2O2, HOCl, chloramine and peroxynitrite (ONOO-, an (p < 0.001). Also, COPD patients with exacerbation had effector molecule of nitric oxide radical, NO), while 7,8- significantly higher (p < 0.001) serum and sputum neopterin dihydroneopterin is mainly acting as a scavenger [17,18]. levels (23.47 ± 2.56 nmol/l and 39.94 ± 5.47 nmol/l respec- Neopterin derivates seem to be able to also interfere with tively) in comparison with stable COPD patients (18.69 molecular biological pathways that are regulated by ± 4.76 nmol/l and 27.91 ± 5.95 nmol/l respectively). Other redox-balances in general. In vitro, derivatives of neopterin workers found similar results. Takabatake and his colleagues induce expression of the nuclear factor-jB [19–21] and of the Role of neopterin among COPD patients 27 iNOS-gene [22]. Similar to this, programed cell death, apopto- [12] N. Takabatake, M. Sata, S. Abe, S. Inoue, H. Saito, H. Yuki, Y. sis, is induced or intensified by neopterin derivates [23–27]. Shibata, I. Kubota, Impaired systemic cell-mediated immunity These findings suggest a new role for neopterin in the and increased susceptibility to acute respiratory tract infections pathogenesis of COPD. Previous reports show that neopterin in patients with COPD, Respir. Med. 99 (4) (2005) 485–492. and 7,8-dihydroneopterin upregulate expression of proto- [13] G. Warwick, P.S. Thomas, D.H. Yates, Non-invasive biomarkers in exacerbations of obstructive lung disease, oncogene c-fos, so these substances may be even involved in Respirology 18 (5) (2013) 874–884. malignant transformation of cells [28,29]. [14] R. Garrod, J. Marshall, E. Barley, S. Fredericks, G. Hagan, The relationship between inflammatory markers and disability in Conclusions chronic obstructive pulmonary disease (COPD), Prim. Care Respir. J. 16 (4) (2007) 236–240. This study found increased neopterin serum and sputum levels [15] V.B. Djordjevic, I. Stojanovic, V. Cosic, L. Zvezdanovic, M. Deljanin-Ilic, S. Dimic, B. Kundalic, T. 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