“Role of Non-Invasive Urinary Biomarkers 3-Bromotyrosine and Leukotriene E4 in the Assessment of Levels of Asthma Control and Predicting Its Flare-up in Children”

“Role of Non-Invasive Urinary Biomarkers 3-Bromotyrosine and Leukotriene E4 in the Assessment of Levels of Asthma Control and Predicting Its Flare-up in Children” | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 29 May 2025 V1 Latest version Share on “Role of Non-Invasive Urinary Biomarkers 3-Bromotyrosine and Leukotriene E4 in the Assessment of Levels of Asthma Control and Predicting Its Flare-up in Children” Authors : Nitesh Gupta R , Krishna Yadav 0000-0001-8900-7441 [email protected] , Sunita Yadav , Manish Raj Kulshrestha , Dipti Agarwal , and LALIT TAKIA Authors Info & Affiliations https://doi.org/10.22541/au.174853995.51881787/v1 271 views 141 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: To see the levels of non-invasive urine markers, such as 3-bromotyrosine and LTE4, in children with asthma and their associations with different degrees of asthma controls and flare-up. Methods: This was a prospective observational study with case control design where cases were twice in numbers in comparison to controls. The included were children aged between 3 to 15 years diagnosed with asthma, as per the GINA guidelines 2022 through outdoor and indoor services. Written informed consent was obtained from parents or legal guardians prior to enrolment. The ethical clearance was taken prior to the start of recruitment of the study subjects from the Institute’s Ethical Committee. Quantitative estimation of urinary 3-bromotyrosine and leukotriene E4 levels was conducted using liquid chromatography-mass spectrometry. Results: There were 146 children with various stages of asthma (asthma group), and 73 healthy children (control group). The asthma group exhibited significantly higher levels (p=<0.001) of urinary 3-bromotyrosine (22.35±18.5 ng/mg creatinine) when compared to healthy controls (14.5±11.2 ng/mg creatinine). Urinary 3-bromotyrosine also showed statistically significant (p=0.025) higher levels in children with flare-up (25.14±19.76 ng/mg creatinine) when compared to well-controlled (18.16±15.48 ng/mg creatinine). After controlling symptoms in hospitalized children due to flare-up, there was significant decrease in the levels of urinary 3-bromotyrosine. Similarly, Urinary LTE4 was significant higher (p=<0.001) in children of asthma group (342±151pg/mg creatinine) in comparison to healthy controls (84.5±38.2pg/mg creatinine). There were statistically significant differences of urinary LTE4 levels between well-controlled and flare-up. Urinary LTE4 also decreases after controlling symptoms who were hospitalized due to flare-up. The urinary levels of 3-bromotyrosine and LTE4 were showed similar rising and decreasing pattern across various subgroups of asthma viz well-controlled and flare -up. The Pearson’s correlation coefficient between 3-bromotyrosine and LTE4 was r=0.214 with a ‘p’ value of 0.009. The ROC curve of 3-bromotyrosine and LTE4 for predicting flare-ups showed that both are moderately able to predict asthma flare-up. Conclusion: LTE4 and 3-bromotyrosine are non-invasive urinary biomarkers were higher in uncontrolled asthma and flare-up. Both had similar trend of increasing or decreasing in various levels of control of asthma and flare-up. The area under curve was higher for LTE4. Introduction: The most widely recognized recommendation in the field of pediatric asthma is Global Initiative for Asthma (GINA) guidelines [1]. These recommendations stress the need to reduce airway inflammation in addition to controlling asthma. This is because numerous studies have shown that airway inflammation directly causes airway obstruction and hyperresponsiveness, which results in clinical symptoms both at baseline and, worse, during flare-up [1, 2]. Therefore, eliminating or reducing airway inflammations is essential for the best management of asthma. Underlying airway inflammations can be estimated using intrusive techniques, which are frequently challenging to execute on children, particularly the younger ones. Therefore, non-invasive markers are need of hours to get tested for their utility and accuracy. Mechanism-based non-invasive markers that concurrently monitor the level of asthma control, the degree of underlying airway inflammations, and the risk of future flare-up may prove to be highly beneficial in the clinical settings to achieve the goals specified in the GINA guidelines. Sputum eosinophils have shown some promise in this regard. They serve as an indirect metric of the extent of eosinophilic airway inflammations that, according to early studies, tracks with indexes of asthma control and maybe even predicts flare-up in children with asthma [3, 4]. Unfortunately, the hypertonic saline solution induction protocols are not well accepted, making it somewhat difficult to get samples for sputum eosinophils, especially in youngsters. In addition, the necessary cytologic testing is time-consuming and rather costly. Exhaled nitric oxide (eNO) is a different and possibly more tolerable non-invasive diagnostic tool for tracking airway inflammation. According to certain research, eNO levels and eosinophilic airway inflammations are related [2]. Nevertheless, eNO also has drawbacks, such as an ambiguous correlation with the likelihood of an asthma attack [5, 6]. In addition to eNO, a wide variety of oxidant species are produced by the inflammatory environment of asthmatic airways [7]. Eosinophils upon recruitment to asthmatic airways and activation by proinflammatory mediators produce strong brominating oxidants, including hypobromous acid and release granule proteins, such as eosinophil peroxidase, which produce a respiratory burst [8, 9]. In order to create reactive brominating species, a powerful antimicrobial oxidant that can preferentially brominate protein tyrosine residues to form 3-bromotyrosine, eosinophil peroxidase uses respiratory burst-generated hydrogen peroxide [8, 9] . As a reasonably specific molecular indicator of eosinophil activation, 3-bromotyrosine can be tested non-invasively in urine as it is a stable molecule. Urinary 3- bromotyrosine is actually a non-invasive marker that is higher in asthma patients and linked to several spirometry related measures of airway blockage in a recent clinical trial involving patients with asthma and healthy control subjects [10]. Cysteinyl leukotrienes (cys-LTs) C4, D4, and E4 play an important role in asthma pathogenesis [11, 12]. They increase bronchial hyperreactivity and constrict bronchial smooth muscle [13]. LTC4 metabolite is highly unstable and rapidly transformed into LTD4, which is then further transformed into a less potent LTE4 which is the most stable of the three metabolites and is eliminated in the urine [14, 15]. Therefore, assessment of urinary levels of LTE4 can be done to evaluate changes in the rate of total body cys-LTs production [14]. Non-invasive indicators such as urinary LTE4 levels have a lot of potential for assessing inflammation in children with asthma [11, 15]. Asthma patients had considerably greater urinary LTE4 levels than controls, according to a number of studies [13, 15, 16]. Urinary LTE4 levels during acute flare-up and their relationship to total serum IgE levels, however, have been the subject of very few research [11]. Therefore, in addition to total IgE levels and absolute eosinophil counts, the current study is investigating the levels of non-invasive urine markers, such as 3-bromotyrosine and LTE4, in children with asthma and their associations with different degrees of asthma controls and flare-up. Methods: This was a prospective observational study conducted from January 2023 to June 2024 at the Department of Pediatrics and the Department of Biochemistry, Dr. R.M.L. Institute of Medical Sciences, Lucknow. The included were children aged between 3 to 15 years diagnosed with asthma, as per the GINA guidelines 2022 through outdoor and indoor services. The level of asthma control was classified as well-controlled, partly controlled, or uncontrolled, and the severity of flare-up was categorized as mild, moderate, severe, or imminent respiratory failure. Written informed consent was obtained from parents or legal guardians prior to enrollment. Additionally, assent was obtained from children aged 7 years and above, in accordance with ethical guidelines. Excluded were children with not fulfilling the criteria of asthma as per GINA guidelines, not willing to participate in the study, pneumonia, tuberculosis, suspected or proven cystic fibrosis, primary ciliary dyskinesia and congenital or acquired heart disease, and on systemic steroid or any other immunosuppression. The required sample size was calculated using the formula: n = z 2 p(1-p)/e 2 , where z is 1.96 (for a 95% confidence interval), is the prevalence of asthma in children (5% based on the ISAAC study [17] for the Indian subcontinent), and is the margin of error (0.05). The calculated sample size was 219 children. Among 219 children, 146 were asthmatics and 73 were age sex matched healthy children. The ethical clearance was taken prior to the start of recruitment of the study subjects from the Dr R. M. L. Institute of Medical Sciences’ Ethical Committee (IEC No. 24/23). After obtaining informed written consent, spot clean-catch urine samples of 10 ml were taken at the initial study encounter. In hospitalized children in flare-up, the repeat urine samples were obtained after controlling their symptoms typically between 3 to 5 days of hospitalization. The collected samples were transported to the Department of Biochemistry on the same day and stored at −20°C till further laboratory procedures. Laboratory estimations of biomarkers were performed fortnightly or sooner if sufficient samples were collected. A venous blood sampling was also done aseptically to withdraw 2 ml of whole blood in each, plain and EDTA vials. These samples were processed in laboratory for hemogram, serum creatinine, absolute eosinophil counts and total IgE levels. Quantitative estimation of urinary 3-bromotyrosine and LTE4 levels was conducted using liquid chromatography-mass spectrometry (LC-MS/MS), a technique validated through internal quality control checks and comparison with standard reference methodologies. The LC-MS/MS was done on QTRAP 5500+, AB Sciex, USA. Standardisation of 3-bromotyrosine and LTE4 done by concurrently measured urine creatinine to account for differences in hydration. Urinary creatine was measured by modified Jaffe’s method on Beckman Coulter AU480, USA by using commercial kits from the same manufacturer as instructed in kit insert. Demographic variables (age, sex, weight, height, socioeconomic status, immunization status, nutritional status), clinical parameters (asthma control level, flare-up severity, medication usage), and laboratory variables (absolute eosinophil counts, total IgE, urinary 3-bromotyrosine, and LTE4 levels) were recorded using pre-designed questionnaires, which were validated for reliability and consistency through a pilot test conducted on a subset of 15 participants prior to the main study. Radiological (Xray-Chest) interpretation was done by one of radiologists at the study center. Data were analyzed using appropriate statistical tests to determine correlations between urinary biomarkers and asthma control, flare-up severity, and total serum IgE levels. Continuous variables are shown as Mean ± SD and categorical variables as number and percentage. For comparing continuous data, student ‘t’ test with two tail value and categorical data, chi (χ 2 ) square tests are used. A ‘p’ value below 0.05 are considered as significant. Pearson’s correlation coefficients were used for association studies, with significance set at p<0.05. Area under curve (AUC) was calculated for both urinary biomarkers. jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Fig1. Flow of the study Results The present study provides a comprehensive analysis of the demographic, clinical, and biochemical characteristics of children with asthma, comparing them to a healthy control group of the children, and offers several important insights into the disease profile and management. Of the 228 children with asthma who were screened, 82 were excluded either because they were unwilling to participate in the study (n=41), had suspected or confirmed cystic fibrosis (n=9), were on systemic steroids (n=9), had concurrent pneumonia (n=13), or had congenital heart disease (n=10) and 146 children with various stages of asthma (asthma group) were recruited. The controls were 73 healthy children (control group) recruited from immunization clinic. The study flow is given as Fig.1. Demographically, the asthma group was predominantly male (61.64%) and largely comprised children aged 5-10 years (64.4%), with a mean age of 7.69±2.6 years. On comparing demographic characteristics like distribution of gender, age, parent’s education level, family income, and immunization status for their age, the control group has similar findings without statistical differences. Clinically, the majority of asthma children were well-controlled (65.18%), though a significant proportion (23.28%) experienced flare-up. Radiological findings indicated that 50% of asthma patients had normal chest X-rays, while 26.72% showed hyperinflation and 19.18% exhibited bronchial wall thickening, consistent with typical asthma-related changes. Anthropometric measures, including weight, height, weight for age, weight for height, height for age and BMI, did not significantly differ between the asthma and control groups, suggesting that these two groups of children were comparable. However, the prevalence of undernutrition, wasting and stunting was quite high in the both groups point to a higher frequency of these conditions in children in this region. The prevalence of moderate undernutrition, wasting and stunting among children of asthma group were 24.7%, 14.4% and 19.2%, respectively while severe undernutrition, wasting and stunting among children of asthma group were 8.2%, 5.5% and 11.6%, respectively. Similarly, the prevalence of moderate undernutrition, wasting and stunting among healthy children of control group were 23.3%, 13.7% and 17.8%, respectively while severe undernutrition, wasting and stunting among children of asthma group were 8.2%, 5.5% and 10.9%, respectively. These findings are in accordance to the observations of national family health survey (NFHS-5, 2019-21) [18]. NFHS-5 data showed prevalence of stunting 35.5%, wasting 19.3% and underweight 32.1%. Among common laboratory parameters including Hb levels, total leucocyte counts, serum bilirubin, SGPT, SGOT, serum urea and creatinine were similar without statistical significance (table 1). The present study demonstrated that absolute eosinophil counts, total IgE, Neutrophil/Eosinophil ratio and Neutrophil/Lymphocytes ratio were statistically significantly higher in children of asthma group in comparison to healthy children. These were also significant when compared between well-controlled asthma and flare-up. However, these characteristics were not significant when were compared between well-controlled and other children of asthma group (table 1). In the present study, the asthma group exhibited significantly higher levels (p=<0.001) of urinary 3-bromotyrosine (22.35±18.5 ng/mg creatinine) when compared to healthy controls (14.5±11.2 ng/mg creatinine) (table 2). Urinary 3-bromotyrosine in asthma group were significant lower when compared other children of asthma group (table 3). Urinary 3-bromotyrosine also showed statistically significant (p=0.025) higher levels in children with flare-up (25.14±19.76 ng/mg creatinine) when compared to well-controlled (18.16±15.48 ng/mg creatinine) (table 4). After controlling symptoms in hospitalized children due to flare-up, there was significant decrease in the levels of urinary 3-bromotyrosine (table 5). Similarly, Urinary LTE4 was significant higher (p=<0.001) in children of asthma group (342±151pg/mg creatinine) in comparison to healthy controls (84.5±38.2pg/mg creatinine) (table 2). However, LTE4 does not show any difference on comparing between well-controlled asthma and other children of asthma group (table 3). Although, there were statistically significant differences of urinary LTE4 levels between well-controlled and flare-up (table 5). Urinary LTE4 also decreases after controlling symptoms who were hospitalized due to flare-up. The urinary levels of 3-bromotyrosine and LTE4 were showed similar rising and decreasing pattern across various subgroups of asthma viz well-controlled and flare-up. The Pearson’s correlation coefficient between 3-bromotyrosine and LTE4 was r=0.214 with a ‘p’ value of 0.009 (fig. 2). The Pearson’s correlation coefficients between these biomarkers and absolute eosinophil counts and total IgE were not significant. The ROC curve of 3-bromotyrosine and LTE4 for predicting flare-up showed that both are moderately able to predict asthma flare-up. The LTE4 has higher area under curve than 3-bromotyrosine (fig. 3). Table 1: Laboratory characteristics of Asthma group and healthy controls Laboratory characteristic Asthma group (Mean ± SD) (a) Healthy controls (Mean ± SD) (b) p-value (a vs b) Well-control Asthma (Mean ± SD) (c) Flare-up (Mean ± SD) (d) p-value (c vs d) Hb 11.9±2.5 12.5±2.8 0.109 10.9±0.84 10.98±0.97 0.738 TLC 13356.5± 6914 13242± 5242 0.900 13502±5410 12573.94± 5548.25 0.414 Absolute Eosinophil Count (AEC) 334±145.5 165.4±124.2 <0.001 315±132.2 386.2±161.46 0.017 Total IgE (IU/ml) 535.5±190.3 122.4±35.8 <0.001 478.6±168.1 585.4±215.7 0.006 Neutrophil/Eosinophil (NER) 23.5±3.2 21.5±3.9 <0.001 22.32±3.2 24.16±3.24 0.007 Neutrophil/Lymphocytes (NLR) 3.42±3.4 1.85±1.6 <0.001 1.75±1.2 2.55±1.43 0.003 S. bilirubin 0.52±0.35 0.47±0.35 0.320 0.44±0.34 0.42±0.38 0.785 SGOT 31.39±7.2 30.40±6.0 0.312 31.53±6.3 30.83±6.89 0.604 SGPT 27.3±8.1 28.8±5.1 0.150 27.3±7.7 26.73±7.92 0.724 Serum Urea 28.74±12.5 30.4±10.7 0.332 29.10±12.5 29.06±13.99 0.988 Serum Creatinine 0.52±0.20 0.47±0.31 0.151 0.47±0.23 0.48±0.21 0.830 jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Table 2: Urinary 3-bromotyrosine and LTE4 in children with asthma and healthy controls Urinary 3-bromotyrosine (ng/mg creatinine) Mean ± SD 22.55±18.5 14.5±11.2 <0.001 Urinary LTE4 (pg/mg creatinine) Mean ± SD 342.0±151 84.5±38.2 <0.001 Table 3: Urinary 3-bromotyrosine and LTE4 in well-controlled Asthma and other children of Asthma group Urinary 3-bromotyrosine (ng/mg creatinine) Mean ± SD 18.16±15.48 21.29±18.3 0.004 Urinary LTE4 (pg/mg creatinine) Mean ± SD 295.0±145 321.2±148 0.371 * Other children of asthma group= asthma group (n=146) – well-controlled asthma (n=73) Table 4: Urinary 3-bromotyrosine and LTE4 in children with well-controlled Asthma and Flare-up Urinary 3-bromotyrosine (ng/mg creatinine) Mean ± SD 18.16±15.48 25.14±19.76 0.025 Urinary LTE4 (pg/mg creatinine) Mean ± SD 295.0±145 435.5±195 <0.001 jabbrv-ltwa-all.ldf jabbrv-ltwa-en.ldf Table 5: Comparison of Urinary 3-bromotyrosine and LTE4 in Flare-up at admission and after symptom control Urinary 3-bromotyrosine (ng/mg creatinine) Mean ± SD 25.14±19.76 20.32 ± 15.27 0.033 Urinary LTE4 (pg/mg creatinine) Mean ± SD 435.5±195 325.8±152.3 0.011 Fig 2: Pearson correlation between 3-bromotyrosine and LTE4 After plotting a line fit plot taking urinary values of 3-bromotyrosine at X-axis and LTE4 at Y-axis, the results of the Pearson correlation indicated that there is a significant small positive relationship between X and Y, ( r = .214, p = 0.009). Fig 3: ROC curve comparing 3-bromotyrosine and LTE4 for predicting flare-up (green curve represents urinary 3-bromotyrosine, red curve represents urinary LTE4, and grey dashed line represents random prediction). The AUC values indicate how well each biomarker distinguishes between flare-up and well-controlled asthma groups. Higher AUC values signify better predictive performance. Discussion GINA guidelines [1], the most accepted guidelines globally, in the field of asthma in children emphasizes for a reduction of airway inflammation because it directly contributes to airway hyperresponsiveness and obstruction [1, 2]. Therefore, for best control of asthma, it is mandatory to abolish or minimize airway inflammation. Over the past few decades, asthma has become more common worldwide. Those who have the condition run the risk of experiencing severe flare-up, necessitate emergency care [16]. Predicting flare-up and reducing their frequency are critical for the management of asthma. Generally, an effective treatment option often available but severe flare-up are difficult to treat and potentially life threatening. It causes significant morbidity and healthcare costs. There is a great need for effective and specific biomarkers for asthma severity, flare-up and response to treatment. There are invasive methods to estimate under lying airway inflammation which are often difficult to perform in children. Hence, non-invasive markers are need of hours to get tested for their utility and accuracy. In order to meet the objectives outlined in the GINA guidelines, mechanism-based non-invasive markers that simultaneously track the degree of underlying airway inflammation, the degree of asthma control, and the risk of future flare-up may prove to be extremely useful in the clinical settings. In the present study, authors have studied two non-invasive biomarkers, 3-bromotyrosine and LTE4, in children suffering from asthma. The 3-bromotyrosine is a stable and reasonably specific molecular indicator of eosinophilic activation [8, 9]. Cysteinyl leukotrienes (cys-LTs) C4, D4, and E4 play an important role in asthma pathogenesis [11, 12]. The LTE4 is the most stable of the three metabolites and is eliminated in the urine [14, 15]. It serves as a potential marker of leukotriene driven inflammation along with eosinophilic inflammation in asthma. It is particularly useful in NSAID-exacerbated asthma [11, 16]. Its high level could be a marker of poor asthma control, helps in guiding to avoidance of NSAIDS and benefitted by use of leukotriene receptor antagonists [13, 15]. The studies related to assessment of urinary biomarkers provided valuable insights into asthma-related inflammation. The LTE4, was significantly elevated in asthma patients compared to healthy controls (p<0.001) in the present study. This finding is consistent with the established role of leukotrienes in promoting bronchoconstriction, airway inflammation, and mucus production in asthma. Notably, LTE4 levels were significantly higher during asthma flare-up compared to well-controlled asthma patients (p<0.001), reinforcing its potential utility as a non-invasive biomarker for monitoring poor disease control and predicting flare-up. The ability to measure LTE4 levels through a stress-free urine collection specially in children could enhance the clinical assessment of asthma control, providing an objective measure that complements traditional symptom-based evaluation. In addition, urinary 3-bromotyrosine, a marker associated with oxidative stress and eosinophilic inflammation, was also significantly higher in asthma patients compared to healthy controls (p<0.001). Its levels differ significantly (p=0.025) between well-controlled and flare-up groups, suggesting its efficacy to assess asthma control. These findings indicate that 3-bromotyrosine may be a useful marker for identifying asthma and monitoring disease control. The identification of urinary LTE4 and 3-bromotyrosine as reliable, non-invasive biomarkers for assessing asthma severity, poor disease control and monitoring flare-up is particularly significant. The markedly elevated levels of LTE4 and 3-bromotyrosine observed during asthma flare-up compared to well-controlled suggest its potential utility as a predictive and diagnostic tool for acute flare-up. Given these findings, larger longitudinal studies are recommended to validate these biomarkers’ clinical usefulness and explore their potential incorporation into standard asthma monitoring protocols. To the best of our knowledge, there are very limited studies on urinary 3-bromotyrosine and LTE4 and its correlation with various level of outcome control and flare-up. Moreover, these studies are done on adult population. Therefore, present study provides very useful insights which is also a merit of the current study. The findings of present study highlight the multifactorial nature of asthma, where demographic, socio-economic, and inflammatory factors collectively contribute to disease manifestation and severity. The substantial differences observed in laboratory markers, particularly LTE4 and 3-bromotyrosine, emphasize the potential of these biomarkers for use in clinical practice to aid in disease monitoring and management. Furthermore, the observation that anthropometric characteristics do not differ significantly between asthmatic and healthy controls suggests that asthma, particularly when well-controlled, may not adversely affect growth and nutritional status. Moreover, the present study found no significant associations between asthma prevalence and socioeconomic factors, including family income, parental education, or immunization status. These observations suggest that socioeconomic status did not play a measurable role in asthma prevalence within this study population. Additionally, the similar immunization rates observed between asthma and control groups indicate adequate vaccination coverage, which might not significantly influence asthma management or exacerbate asthma symptoms in this particular context. Future research could further explore whether different socioeconomic contexts or larger populations reveal associations not evident in this study. The findings of present study largely align with existing literature concerning demographic, socio-economic and biomarkers characteristics associated with asthma in children. The demographic profile in this study, characterized by a predominance of males (61.64%) and children aged between 5–10 years (64.4%), is consistent with previous studies by BJ Arun et al. (2015) [19] and Rao S et al. (2011) [20], which reported similar demographic characteristics. Although socio-economic factors such as family income and parental education were examined, present study did not find statistically significant differences between the asthma and control groups, suggesting these factors might not substantially impact asthma prevalence or severity within this population. Similarly, immunization status did not significantly differ between groups, indicating adequate vaccination coverage in both groups. Anthropometric measurements also showed no significant differences, supporting findings by Krishnankutty C V and Subbaraman K R (2015) [21], who suggested that well-controlled asthma typically does not negatively influence growth or nutritional status. Radiological findings of hyperinflation (26.72%) and bronchial wall thickening (19.18%) align with the structural airway changes commonly reported in asthma literature. Significantly elevated inflammatory markers, including eosinophil counts, total IgE, neutrophil/eosinophil ratio, and neutrophil/lymphocyte ratio, confirm heightened inflammatory activity consistent with existing research by Ban GY et al. (2021) [22]. Importantly, urinary LTE4 levels were significantly elevated during asthma flare-up, underscoring its potential as a non-invasive biomarker for monitoring disease severity and predicting flare-up, which is supported by findings from Di Palmo E et al. (2021) [23]. Similarly, urinary 3-bromotyrosine was significantly elevated during flare-up compared to well-controlled asthma, indicating its potential utility in differentiating asthma severity and highlighting its role as a biomarker of oxidative stress and eosinophilic inflammation as shown by in their studies by Wang Z et al. (2022) [24] and Wedes SH et al. (2011) [25]. Since, urinary 3-bromotyrosine is a biomarker of eosinophil activation and eosinophilic variant of asthma is usually responsive to inhaled corticosteroids unlike to neutrophilic asthma and therefore, higher urinary 3-bromotyrosine indirectly suggestive of responsiveness to inhaled corticosteroids. This has been shown in the a study done by Tiotiu A et al. (2018) [26]. Therefore, higher levels of urinary 3-bromotyrosine are suggestive of poorly controlled though corticosteroids responsive but due to poor adherence to inhaled corticosteroids. Although, urinary 3-bromotyrosine levels are independent biomarker of eosinophil activation in children and not related to absolute eosinophil counts in the sputum and peripheral blood. Similar findings are also shown in the studies by Wu W et al. (2000) [9] and Wedes SH et al. (2011) [25]. In the present study, Pearson’s correlation between absolute eosinophil counts and total IgE with urinary biomarkers 3-bromotyrosine and LTE4 were insignificant. This disconnect between absolute eosinophil counts and their bromination products and total IgE levels is because urinary levels of 3-bromotyrosine reflect the resolution phase of the protein remodelling caused by eosinophilic tissue injury and inflammation. Therefore, measurement of eosinophil counts in sputum and blood are inadequate to assess the oxidative damage and remodelling caused by eosinophilic peroxidase in lung tissues. The present study clearly demonstrates the significant decrease in the urinary levels of 3-bromotyrosine and LTE4 in children hospitalized due to flare-up after controlling their symptoms suggestive of potential marker of disease control. Though results are significant, there is high degree of variability among children with non-severe and severe asthma. This biological variability may limit their use for diagnosis but an individual with known asthma, higher levels of these urinary biomarkers are strongly related to poor control over the disease, flare-up and suggestive of more oxidative stress and tissue remodeling in severe asthma. The range of urinary 3-bromotyrosine and LTE4 in children with asthma varies across different studies, reflecting differences in study design, asthma severity, age groups, and the methods used for measurement. The internal validity of this study is relatively strong due to its prospective observational design. The study’s rigorous methodology, including adherence to well-defined inclusion and exclusion criteria based on GINA guidelines 2022, enhances the accuracy of participant selection and minimizes selection bias. Additionally, the use of structured data collection tools, including validated questionnaires and standardized laboratory protocols for biomarker estimation using LC-MS/MS for LTE4 and 3-bromotyrosine, ensures consistency and reliability of data. The careful classification of asthma control levels and flare-up severity adds further robustness to the findings. Furthermore, measures to ensure quality control in laboratory procedures strengthen the study’s internal validity. The external validity of this study is somewhat limited by certain methodological factors. The study was conducted at a single center which may limit the generalizability of findings to broader populations, especially considering regional differences in asthma prevalence, environmental exposures, healthcare accessibility, and genetic factors. The relatively small sample size, particularly in the control group (n=73), further restricts the representativeness of the study population. Additionally, while the study included children aged 3 to 15 years, it did not account for potential age-related differences in asthma presentation, severity, and biomarker levels. The exclusion of certain conditions (e.g., bronchiolitis, tuberculosis, pneumonia, cystic fibrosis, etc.) ensures a more homogenous sample and the prospective nature of the study improves external validity to great extent by reflecting real-world clinical scenarios and natural disease progression. One of the primary strengths of this study is its comprehensive evaluation of pediatric asthma through an extensive assessment of demographic, socio-economic, clinical, radiological, and biochemical parameters, including inflammatory and urinary biomarkers. This detailed approach offers a well-rounded understanding of asthma pathophysiology and associated factors in children. The inclusion of a matched healthy control group further strengthens the study by facilitating robust comparisons and enhancing the validity of the findings. Additionally, the incorporation of non-invasive urinary biomarkers, such as 3-bromotyrosine and LTE4, provides practical, patient-friendly tools for disease monitoring. In the present study, for estimation of urinary 3-broomotyrosine and LTE4, the state of art cutting edge technology namely LCMS-MS has been used. The significant elevation of LTE4 during asthma flare-up supports its potential as a reliable biomarker for predicting and assessing asthma severity. Furthermore, the findings regarding elevated 3-bromotyrosine levels during flare-up suggest its utility in identifying disease control, chances of flare-up and its potential role as an additional non-invasive marker in clinical practice. Overall, the study’s comprehensive methodology, strict adherence to study protocol and laboratory procedures, and the identification of clinically useful biomarkers align well with existing literature, strengthening its relevance and applicability. The results of the present study might have a great impact on routine practice and care of children with asthma in the monitoring of disease control, progression, flare-up and compliance towards treatment. Best of our knowledge this is very first study which is assessing two non-invasive urinary biomarkers simultaneously in children with asthma and correlation between these markers also. Despite the strengths of the present study, there are few limitations should be acknowledged. Its observational design limits the ability to establish causal relationships between the observed variables and asthma outcomes. The reliance on single-time measurements of biomarkers (urinary LTE4 and 3-bromotyrosine) at baseline and follow-up visits might not fully capture their dynamic fluctuations related to asthma severity or treatment responsiveness. In the current study follow up sampling done only in children hospitalized due to flare-up after controlling their symptoms typically within 3 to 5 days. It would have better if follow-up period would have been longer and for all cases to predict flare-up. The classification of asthma control and severity based on patient-reported symptoms and clinical evaluations, without objective measurements such as spirometry, may introduce bias and limit the accuracy of these categorizations. Therefore, further large-scale, multicentric, longitudinal studies incorporating repeated biomarker measurements and objective clinical assessments are recommended to validate the clinical applicability and reliability of these biomarkers. Since, there are several phenotypes of wheezing below 3 years therefore age group of 3 and above only up-to 15 years were considered in the present study. However, spirometry could not be done in the present study as many of recruited subjects would be unable to do spirometry. In the present study, sputum samples for estimation of eosinophil counts and its correlation with urinary biomarkers were not done. However, blood eosinophil count estimation was done. There are many studies demonstrating no correlation between sputum eosinophil count with flare-up and severity or levels of control of the illness [5, 6]. It is difficult to take sputum sample in children. Quality of samples are also an issue along with its microscopic examination has been manpower consuming and time taking along with high inter-observer variation. 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Keywords 3-bromotyrosine asthma children leukotriene e4 urinary biomarkers Authors Affiliations Nitesh Gupta R Dr Ram Manohar Lohia Institute of Medical Sciences View all articles by this author Krishna Yadav 0000-0001-8900-7441 [email protected] Dr Ram Manohar Lohia Institute of Medical Sciences View all articles by this author Sunita Yadav Hind Institute of Medical Sciences View all articles by this author Manish Raj Kulshrestha Dr Ram Manohar Lohia Institute of Medical Sciences View all articles by this author Dipti Agarwal Dr Ram Manohar Lohia Institute of Medical Sciences View all articles by this author LALIT TAKIA All India Institute of Medical Sciences New Delhi View all articles by this author Metrics & Citations Metrics Article Usage 271 views 141 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Nitesh Gupta R, Krishna Yadav, Sunita Yadav, et al. “Role of Non-Invasive Urinary Biomarkers 3-Bromotyrosine and Leukotriene E4 in the Assessment of Levels of Asthma Control and Predicting Its Flare-up in Children”. 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