Human Neutrophil Gelatinase-Associated Lipocalin: A New Biomarker in Asthma-COPD Overlap Syndrome

Human neutrophil gelatinase-associated lipocalin (NGAL) is a protein considered as a noninvasive prognostic biomarker in Asthma-COPD overlap syndrome (ACO). In this study we aimed to show the usability of NGAL as a biomarker in ACO characterized with airway inammation. MATERIAL-METHOD: Three patient groups (Group I: COPD, n: 50, Group II: Asthma, n:50, Group III: ACO, n:50) and a healhty non smoker group (Group IV, n:50) are taken into study. The serum NGAL levels of groups are compared. In addition, in Group I,II,III the level of serum NGAL is compared with the age, duration of smoking (pocket/year), number of attack per year, respiratory function values, serum C reaktive protein (CRP, mg/dL), serum IgE (IU/mL), serum neutrophil (%) and serum lymphocyte (%), blood neutrophil-to-lymphocyte rate and atopy. inammation


Introduction
Asthma is a heteregenous chronic airway diseases which leads to reversible air ow obstruction based on innate and adaptive immune responses. Heterogenity of asthma is due to genetic and environmental reasons, chronic aiway diseases affecting 1-18% of the population (1). In the majority of patients are underlying atopy. In ammatory cell-derived mediators cause increased vascular permeability, mucosal edema, bronchial smooth muscle hypertrophy and subepithelial brosis (2)(3)(4)(5)(6). Chronic obstructive pulmonary disease (COPD); It is a disease characterized by progressive air ow restriction that is not fully reversible. This disease develops as a result of an in ammatory process against harmful gases and particles, especially cigarette smoke. In ammation is not only limited to the lungs, but also shows Page 3/17 systemic features (7). COPD, which is a preventable and treatable disease, progresses with exacerbations that increase in severity and frequency. Chronic airway obstruction in COPD develops due to narrowing of small airways and parenchymal destruction. Chronic in ammation causes structural changes in the small airways. This in ammatory process and parenchymal destruction lead to loss of alveoli and a decrease in the elastic return pressure of the lungs (8). Asthma-COPD overlap (ACO) refers to a group of poorly studied and characterised patients reporting with disease presentations of both asthma and COPD, thereby making both diagnosis and treatment challenging for the clinicians (9)(10)(11). ACO can be de ned as symptoms accompanying increased airway sensitivity, reversible airway obstruction and persistent air ow restriction. While at present there is no general consensus on de nition or de ning features for this subgroup of patients, there is broad agreement that they experience frequent exacerbations (12). The prevalence of ACO is 15-55% in the population (13). They exhibit a higher burden in terms of both mortality and morbidity in comparison to patients with only asthma or COPD. The pathophysiology of the disease and its existence as a unique disease entity remains unclear (11).
Neutrophil Gelatinase-associated Lipocalin (NGAL), also known as neutrophil glucosaminidaseassociated lipocalin, is a 178 amino acid, 25 kDa protein synthesized in neutrophils and epithelial cells (14). Gene expression has been demonstrated in the uterus, prostate, salivary glands, lung, trachea, stomach, colon and kidney (15,16). Neutrophil gelatinase-related lipocalin production increases in humans in response to ACO and can be detected in serum at a very early stage.
In recent years, in studies conducted in ACO patients, there are limited numbers of publications about the high serum lipocalin levels associated with human neutrophil gelatinosis and that it is associated with the severity of the disease. This study was planned considering that there is a limited amount of literature information in association with NGAL and asthma, COPD and ACO and it should be supported by new studies on this subject.

Patients
Hundred and sixty patients aged 18 to 65 years who were diagnosed with ACO, COPD and asthma according to the GOLD, GINA 2019 criteria between May 2016 and December 2020, which was done at the Diskapı Yildirim Beyazit Trainig and Research Hospital Chest Diseases Clinic, Ankara Turkey. However, the full article has not previously been published. Three patient groups (Group I: COPD, n: 40, Group II: Asthma, n: 40), Group III: ACO, n:40) and a healhty non smoker group (Group IV, n:40) are taken into study.
Asthma, ACO and COPD was excluded in the control group by demographic data, physical examination and respiratory function tests. Detailed physicals exams, detailed blood tests, height, weight and neck circumference measurements were conducted. The body mass index (BMI, kg/m2) of each patient was calculated by dividing the body weight in kilograms by the square of the patient's height in meters. Assuming its bene t in exclusion of the respiratory system pathologies, posteroanterior chest radiography was performed. Pulmonary function tests were performed in the respiratory laboratory of our hospital, using a Jaeger brand spirometer according to the criteria of American Thoracic Society (ATS).
Forced expiratory volume in one second (FEV1, liters), and forced vital capacity (FVC, liters), and FEV1 to FVC (%) values are measured at least 3 times, and optimal values were recorded.
Twentyfour of well controlled asthmatic patients were categorized as mild persistant, 16 of them as moderate persistant according to GINA before treatment. The patients also underwent physical examinations. One or more positive results to allergens in skin prick test (Allergopharma, Germany) was accepted as atopy. According to previous skin prick test results of patients regullary followed in our clinics, 9 asthma patients (3 mild peristant, 6 moderate persistant) and 3 ACO patients had atopy.
Patient with ACO was categorized according to GINA and GOLD criteria (17). The treatment of our patient's protocols was explained to GOLD and GINA criteria. Anemia was de ned as Hb < 12 g/dl in male.
Baseline arterial oxygen saturation was de ned ≤ 88%. Our exclusion criteria were an acute COPD, ACO and exacerbations in the last 3 months (increased cough, dyspnea, sputum production, and/or purulence), other acute and chronic diseases, a blood transfusion in the last 6 months, anti-in ammatory therapy (oral, parenteral systemic glucocorticosteroids) within the last 3 months, and anemia within the last 6 months, malignancy and pregnancy. The study was planned in accordance with the suggestions of the Helsinki Document and Dışkapı Yıldırım Beyazıt Research and Education Hospital's ethic commission. Signed consent forms were obtained from all the patients who volunteered.

Laboratory
In all patient groups and control group included in the study, blood and serum samples for full blood testing were taken in the morning on an empty stomach. Serum CRP (0-6 mg/L ), serum Ig E (0-180 IU/mL), serum neutrophil (%), lymphocyte (%) levels were assessed using standard laboratory methods. The blood sample drawn for the measurement of NGAL were collected early in the morning after a minimum of 10 hours fasting and centrifuged for 10 min at 3000 rpm, and serum was stored at -80 C in aliquots until the day of analysis. Serum NGAL levels were measured using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA), according to the instructions of the producer company (Hangzhou Eastbiopharm Co. Ltd., Hangzhou, China). Results were given as ng/mL. The intra-assay coe cient of variation (CV) was < 10% and the inter-assay CV was 12%. Chest radiographs were evaluated to exclude other pathologies. The NLR was obtained by dividing the absolute number of neutrophils by the absolute number of lymphocytes. The data were expressed as a percentage value (i.e.: 2.25%).

Statistical Analysis
Statistical analyses were performed using (Predictive Analytics SoftWare) version 20. Descriptive results for numerical variables were expressed as mean ± standard deviation; categorical variables were expressed as frequency and percentage. Chi-square analysis or Fisher's exact test, where appropriate, was used to compare proportions in different groups. There was said to be a statistically signi cant difference between groups when P < 0.005. In that case, difference groups were determined by the Mann-Whitney U test and Bonferroni correction.The relationship of serum NGAL levels with other variables was examined using the Spearman Correlation coe cient.

Results
Demographic and laboratory data for the 160 patients and control group monitored at our clinic were demonstrated in Table 1

Discussion
The aim of this study is to compare and analyse serum NGAL levels at chronic in amatuar lung disease such as asthma, COPD, ACO and to evaluate the clinical utility of serum NGAL as a predictive marker for these disease. In this study, we found that serum NGAL level was higher in ACO group than COPD, asthma and control groups. Moreover, serum NGAL level is increased as the number of attacks is increased (p: 0.021) with ACO patients. On the other hand in the patients having ACO, there was also positive correlation between blood NLR and serum NGAL levels. There was no correlation found among the other variables. Chronic in ammatory lung diseases such as COPD, asthma and ACO are among the commonly seen reasons of death worldwide. Studies have shown that during stable period and exacerbations in patients with chronic in ammatory lung diseases increase both local and systemic in ammatory response. In recent years, serum NGAL in in ammation, endothelial dysfunction, angiogenesis and remodeling have been reported to increases. Until now, NGAL's research continues as an in ammatory marker in the various diesases. Chronic in ammatory lung diseases are one of them.
NGAL is not only attributed to activated neutrophils but could also be secreted by the respiratory epithelial cells in response to in ammatory stimuli and by myeloid and epithelial cells in response to toll-like receptor activation during bacterial infections.
There has been limited number of studies related with NGAL in lung diseases. In rst studies, HNGAL is signi cantly elevated in the bronchoalveolar lavage uid (BALF) and induced sputum of COPD patients (18,19). In normal human lung tissue, NGAL is constitutively expressed within tracheal goblet cells and in type II pneumocytes (20,21). An increase in the level of in ammation occurs with the progression of chronic in ammatory lung diseases. In our study, the detection of increased serum NGAL levels in patients with chronic in ammatory lung diseases in the stable period supported that, rather than being restricted to the lung therefore being local, they are systemic in ammatory diseases.. In previous studies, it was reported that serum NGAL expression increased in various malignancies and it is a prognostic factor in these cancers and can be used in follow-up, after treatment. After acute ischemic and nephrotoxic injury, serum NGAL expression from the renal epithelium increases in response to in ammation (22,23). Serum NGAL exists as an acute phase protein based on high levels in serum, epithelial, urine and fecal levels of patients with active in ammatory disease. There is no su cient data on serum NGAL levels during exacerbation periods in ACO. Our study showed serum NGAL in patients having ACO when the maximum level of serum exist, it is observed that serum NGAL level is increased as the number of attacks is increase.
Bacteria synthesize siderophore. It captures iron from the extracellular space by means of sidedrophores.
Serum NGAL participates in siderophore mediated iron cycle for proliferation and differentiation. Mice lacking serum NGAL gene are more sensitive to some gram (-) bacteria. In addition, mortality rates were found to be higher in sepsis compared to normal mice. Therefore, serum NGAL is de ned as an essential component of innate immunity against bacterial infections. NGAL production increases in bacterial infections and NGAL levels in tissues are used to differentiate bacterial-viral infections. Activated neutrophils secretes matrix metalloprotease 9 (MMP-9). NGAL binds to MMP-9 and prevents prolonged collagen degradation. These actions of NGAL are of particular interest in relation to patients with chronic in ammatory lung diseases especially development of emphysema and airway wall remodeling (24,25). In a study on NGAL, sputum levels of NGAL were signi cantly higher in patients with ACOcompared with subjects without ACO and that level of NGAL was related to important ACO characteristics (26). NGAL is actively secreted from both neutrophils and epithelial cells. The upregulation of NGAL transcription in neutrophils and epithelial cells involves fundamental upstream in ammatory pathways, such as activation of nuclear factor-k B, interleukin-1 activation, and interaction between microbial products and certain toll-like receptors. NGAL may be a marker of several upstream in ammatory pathways, re ecting several processes potentially involved in the pathogenesis of COPD and ACO. In our study, we found that both NLR and exacerbation rate were positively correlated with increasing serum NGAL level, which supports the relationship between increased neutrophil rate and NGAL in in ammation especially patients in ACO.
Systemic in ammation in ACO is similar to COPD. Serum NGAL value was found close to each other in ACO and COPD groups in our study. This result may support the similarity of in ammatory events between COPD and ACO. In another study, the sputum levels of NGAL were signi cantly increased in ACO when compared with COPD and asthma groups. The sputum NGAL levels might be related to airway in ammation and low-grade microbial colonization, which predispose patients with ACO to acute viral infections and exacerbations.
The present study aimed to investigate the potential of NLR, as a prognostic markers in patients with chronic in ammatory lung diseses. It has been demonstrated that NLR may be a useful predictor of systemic in ammation and with prognosis of many cardiovascular diseases, malignancies and chronic in ammatory diseases (27)(28)(29)(30). Taking this idea into account, some studies focused on NLR which are considered among the markers of systemic in ammatory response for COPD, ACO and asthma. One of these have shown that increase in the ratio of circulating blood NLR levels may serve as a marker of systemic in ammation for ACO, COPD patients. The patients with stable disease and healthy controls have shown lower NLR values than the patients with COPD exacerbation, according to the study done by authors in an chronic in ammatory deseases study (31). In a current study were found NLR values increased with restriction of air ow limitation. They tought that the change in NLR was due to COPD itselfs (32). So far, the studies reviewed showed that can be used to predict COPD prognosis and mortality (33). In another study, neutrophils and NLR, as indicators of circulating immune complexes, were found higher in patients with COPD and ACO than those in the healthy population. They thougt that NLR may be used as a biomarker to distinguish and diagnose COPD and ACO (34). With a different perspetcive, in a study was found the level of NLR similar in COPD and ACO patients (35). In another study found that the NLR ratio increased in parallel with the severity of COPD (36). Similar results were found in our study. The NLR ratio increased with the aggravation of in ammation.
Our patient with ACO groups had high serum CRP levels. Ultimately ACO and COPD are associated with systemic in ammation. The increase NLR and CRP may supports this situation (37)(38)(39). Due to the high number of exacerbation period for ACO, we think that the role of in ammatory mechanisms may increase in this desase.
As a result; in patients with chronic in ammatory lung diseases, NGAL may be a systemic useful biomarker re ecting the in ammation level. In additional to chronic systemic in ammation may explain an parellel increase in NLR value, CRP and serum NGAL. This study has some limitations. Additional studies are needed for clinical evaluation of the progression of chronic in ammatory lung diseases. As our study was conducted with a small sample group, the study should be supported with larger scale studies.  Figure 1 shows the box plot graphs of serum NGAL level in all patients groups. Figure 2 shows tho box plot FEV1(L) level of patients and control group.

Figures
Page 15/17 Figure 3 shows the box plot FEV1/FVC (L) level of patients and control group. Forced expiratory volume in one second (FEV1) and FEV1/FVC ratio was higher patients with asthma and control groups. Figure 4 shows the box plot acute exacerbation of patients with Asthma, COPD, ACO. Acute exacerbation was also high in patients with ACO. Relationships between the serum NGAL levels and other variables were demonstrated in table 2. The serum NGAL levels was found signi cantly higher in patients with ACO patients compared to other groups. It was found that this height was positively correlated with number of attack per year (p<0.05). On the other hand in the patients having ACO there was also positive correlation between NLR and serum NGAL (p<0.05). There was no other relation found among the other variables. Figure 5 shows the box plot graphs of the relationship between smoking and serum NGAL level in patients with ACO. Serum NGAL was increasing in proportion to smoking. But not statistically signi cant (p>0.05)