The Role of Serum Myeloperoxidase in Prediction of Severe Bronchial Asthma

Article information Background: Bronchial asthma increasing all over the world and the need of a gold-standard biomarkers for prediction of asthma and follow up of treatment is mandatory. However, it is still not existing. Aim of the work: To measure the serum level of plasma myeloperoxidase [MPO], and its correlation with respiratory function as well as its predictive power of asthma and its severity. Patients and Methods: The study recruited 130 patients with asthma, with age and sex-matched other 130 healthy controls. Patients were clinically evaluated; respiratory functions were performed and blood samples were drawn to estimate the serum levels of MPO. In addition, sputum was assessed for cellular content. Asthma severity was determined according to available guidelines and both mild and moderate forms were considered as [non-severe form of asthma]. Receiver operation characteristic [ROC] curve was built to assess the predictive power of MPO. Results: There was female-sex predominance in the study and control groups [65.4% and 70.0%, respectively]. 15.4% had mild, 35.4% had moderate and 49.2% had severe asthma. MPO levels were significantly higher in the study than the control groups [3310.35±373.39 vs. 1900.32±333.57 pg/ml, respectively]. The serum levels of MPO were positively correlated with asthma severity, all cellular content in BAL and body mass index. However, it was inversely correlated with respiratory function. MPO were significantly higher in severe than non-severe forms [3666.59±736.37 vs. 2964.91±554.47 pg/ml, respectively]. MPO had a good predictive power of asthma and its severe form [the AUC was 0.950 and 0.819 respectively]. Conclusion: Serum levels of MPO can used as a good biomarker for diagnosis of asthma and its severity. But results must be treated with caution, and MPO should be used with a panel of biomarkers till more evidence originated to confirm its role from future studies.

Thus, evaluation of airway inflammation could be investigated by assessment of inflammatory cells or the levels of the serum levels of their mediators. Neutrophil cells contribute to the inflammation process and this contribution is well-known. However, its precise role in bronchial asthma is not well known. Neutrophils release an enzyme known as myeloperoxidase [MPO]. It released from the primary azurophilic granules of the neutrophils [7,8] .
It is elevated in both sputum and blood of asthmatic patients, reflecting the neutrophil activity in BA in both adults and children [9][10][11] . However, its clinical value as a predictor of the development of severe asthma is not well-known.
The current work aimed to measure the serum levels of MPO, as an indicator of neutrophil activity. Then, these values correlated with respiratory function and its ability to predict severe asthma were determined.

Sampling
Blood samples were drawn by venipuncture of the antecubital or any other suitable vein. All samples collected on capillary tubes, centrifuged at 5000 g/min for 15 minutes after coagulation. Eppendorf tubes were used for collection and storage of the serum. Samples were stored at -20 °C till the time of analysis.

Determination of serum myeloperoxidase
The MPO enzyme was measured in the serum samples by MPO, ELISA Kit. Kits were supplied by [Cloud-Clone Corp.®, USA]. The detection of the Kits extends from 78 to 5500 pg/ml.

Total and differential cell counts in BAL
The BAL was collected and saved as pellets, which were resuspended in 200 mL of phosphate buffer solution [PBS]. Hemocytometer was used for cell count by using 50 µl of cell suspension. Another 50 ml of suspension was further subjected to cytospin at 450 rpm for 5 min, followed by Diff-Quick staining to detect the inflammatory cells. A total of 300 cells were counted under microscopic examination. Cells were counted on the basis of its morphological and staining characteristics. These were eosinophils, neutrophils, macrophages or lymphocytes.

Forced spirometry
It was performed according to American Thoracic Society/European Respiratory Society [ERS/ATS] standards [12] .

Statistical analysis
The collected data were anonymized and collected in a tabular form. Then transferred to a personal computer and entered to the statistical package for social sciences, for windows, version 22 [IBM®SPSS®, Chicago, Armonk, USA]. The quantitative data were expressed by their mean and standard deviations, while qualitative data were summarized by their frequency and percentages. Groups were compared by independent samples student "t" test for quantitative variables and by Chi square for qualitative variables. Pearson's correlation coefficient [r] was calculated to determine bivariate correlations between two variables. The receiver operation characteristic [ROC] curve was used to estimate the predictive ability of MPO for severe asthma. Values of area under the curve [AUC] above 0.7 indicate good predictive power. The p value < 0.05 indicates significant results.

RESULTS
In the current work, there was female-sex predominance in the study and control groups [65.4% and 70.0%, respectively] with no significant differences between study and control groups. In the study group, 15

DISCUSSION
Serum MPO was significantly increased with asthma than control groups. In addition, it significantly correlated with respiratory function. When ROC curve constructed to test the predictive power of MPO for asthma and its severe form, it showed a good predictive power [for prediction of asthma, the AUC was 0.950, with sensitivity of 84.62% and specificity of 100.0%]. For sever form of asthma, the AUC was 0.819, with sensitivity of 85.94% and specificity of 86.36% at the MPO values > 3400 pg/ml. These results are in accordance with Aldhalmi et al. [13] who reported significant increase of MPO in asthmatic than healthy controls [3222.5 ± 1280.8 vs. 1670.8 ± 991.6; p value < 0.001]. In addition, they reported a sensitivity of 86.2% and specificity of 86.3% for prediction of asthma. However, the same authors did not find significant differences in the distribution of MPO levels according to asthma levels [mild, moderate and severe forms]. This could be explained by their lower number of included subjects than the current one and abnormal distribution of the MPO levels.
Results of the current work also in line with a previous two trials, that reported significant elevation of serum or sputum levels of MPO in asthma than healthy controls. These results reflecting the neutrophilic effects in asthma and in severe form of asthma [9,14] .
Obaid Abdullah et al. [15] reported that, the mean patients' age was 31.9±15.1 year, with a predominance of females, as in the current work. Mean spirometric parameters were significantly lower among patients than controls [p < 0.001]. MPO was significantly higher among BAs than controls [3222.5±1280.8 vs. 1670.8±991.6; p < 0.001]. However, values of MPO levels have not differed significantly with asthma levels of severity, and correlation with respiratory function was statistically non-significant. ROC curves revealed a sensitivity, specificity and accuracy for MPO [80.9%, 72.1%, and 84.3%], respectively to predict asthmatic severity, indicating good prediction as in the current work.
Wang et al. [16] also reported meaningful variance of MPO levels in asthmatics throughout attacks, but not in asthmatic patients in remission, indicating a role of MPO in pathogenesis and exacerbations of asthma. Also, previous studies recommended that, neutrophils and/or MPO are not the main pathogenic players of asthma inflammation [17,18] ; thus, other roles are suspected. Higher levels of MPO in adult patients with asthma may be related to associated infection, as neutrophil activation defends the body against bacterial invasion. A well-known fact that infections exacerbates the bronchial asthma and this confirm the results of the previous work of Wang et al. [16] .
As the prevalence of bronchial asthma increases all over the world in the last decades, the existence for a gold-standard biomarker is increasingly required. However, and unfortunately, this biomarker is not existing. MPO [according to the results of the current work] seems to be a promising biomarker. However, the current work has some limitations [small number of the included subjects and absence of other biomarkers for comparison]. Besides the contradictory results of the predictive power of MPO reported in previous studies, MPO could not be treated as the golden biomarker in patients with bronchial asthma or its severity. Future studies are recommended. However, it may be included with a panel of biomarkers at the current moment till the emergence of new evidence.