Serum Bilirubin Levels and Disease Severity in Patients with Pneumoconiosis

Aim To investigate the association between serum bilirubin and disease severity in patients with pneumoconiosis. Methods The study comprised 45 patients with pneumoconiosis retrospectively; all pneumoconiosis patients were classified into I, II, and III stage according to the radiological severity. Results Serum direct bilirubin levels were significantly lower in III stage pneumoconiosis patients than those in I/II stage (p = 0.012) but not serum indirect bilirubin. Serum direct bilirubin was negatively correlated with radiological severity in patients with pneumoconiosis (r = −0.320; p = 0.032); by multiple linear-regression analysis, we observed that serum direct bilirubin levels had independent association with radiological severity in patients with pneumoconiosis (beta = −0.459; p = 0.005). Conclusions Serum direct bilirubin levels are negatively associated with disease severity in patients with pneumoconiosis.


Introduction
Pneumoconiosis is a group of heterogeneous occupational interstitial lung diseases caused by long-term accumulation of dust in the lungs [1]. Although many protective measures are taken to confront dust inhalation in workers, however, pneumoconiosis is still prevalent all around the world, and its morbidity has remained at relatively high levels, which seriously threatens global public health [2,3]. Te timely diagnosis and evaluation of pneumoconiosis have close association with the treatment and prognosis in patients with pneumoconiosis; clinically, screening of pneumoconiosis mainly relies on the exposure history of dust and imaging examination [2]. However, few serum biomarkers have been used to evaluate the disease status in patients with pneumoconiosis.
Serum bilirubin, an end metabolic product of heme catabolism, consists of two forms: direct bilirubin and indirect bilirubin [4]. Serum bilirubin has been used as a traditional marker for liver disease in clinical practice [5]. In recent years, serum bilirubin has been reported to be associated with infammatory diseases, such as metabolic syndrome, atherosclerosis, and hypertension [6]. In fact, it is generally believed that bilirubin possesses multiple biological activities including antioxidant and anti-infammatory action [7]. Besides, bilirubin also has cytoprotective properties against oxidative stress [8]. Noteworthy, bilirubin can attenuate pulmonary infammation by its anti-infammatory and antioxidant properties in the rat model of smoke-induced emphysema [9], and antioxidant action of bilirubin can ameliorate bleomycininduced pulmonary fbrosis by inhibiting lung infammation [10]. Pulmonary infammation and oxidative stress have been accepted to be involved with inhalation of dust in the lungs [11,12]. Terefore, we examined the relationship between serum bilirubin and disease severity in patients with pneumoconiosis.

Materials and Methods
Our study included 45 patients with pneumoconiosis retrospectively; all pneumoconiosis patients were diagnosed according to the radiological features and history of occupational exposure. Our study excluded patients with the following conditions: coronary heart disease, hepatic insufciency, renal insufciency, hemolytic disease, gallstone disease, chronic obstructive pulmonary disease, asthma, bronchiectasis, lung abscess, autoimmune disease, and malignant tumors. We used radiological severity to classify pneumoconiosis patients as I, II, and III stage, which is similar compared with the classifcation system of the International Labor Organization criteria [13][14][15]. Te study was approved by the Ethics Committee of Te Afliated Hospital of Youjiang Medical University for Nationalities and conformed to the Declaration of Helsinki.

Statistical
Analysis. Means ± standard deviation or median (interquartile range) was used as descriptive statistics for normal distributed or non-normal continuous variables, respectively. Percentage was used as descriptive statistics for categorical variables. Te Chi-square or Fisher's exact test was used to compare the diferences of categorical variables. Comparative analyses of two continuous variables were performed with Student's t-test or Mann-Whitney U test, when appropriate. Te bivariate correlation was examined by Pearson or Spearman approach appropriately. Multiple linear-regression analysis was further carried out for identifying the independent determinants. All statistical analyses were performed using SPSS 25.0 software version. A p value <0.05 considered as statistically signifcant.

Comparison of Serum Bilirubin Levels among Pneumoconiosis Patients with Diferent Stages.
Te characteristics of patients are described in Table 1. Patients with III stage pneumoconiosis are usually regarded as advanced pneumoconiosis clinically; hence, the diferences of serum direct bilirubin and serum indirect bilirubin were compared between stage I/II and stage III pneumoconiosis patients; we found that serum direct bilirubin levels were signifcantly reduced in III stage pneumoconiosis patients compared with those in I/II stage (p = 0.012) ( Figure 1); serum indirect bilirubin levels have no signifcant diferences between the two groups ( Figure 2). Tere were no signifcant diferences with respect to sex, age, smoking history, pulmonary tuberculosis history, diabetes mellitus, hypertension, total protein, alanine aminotransferase, and aspartate aminotransferase.

Te Correlation Analysis between Serum Bilirubin and Disease Severity in Pneumoconiosis Patients.
Our study estimated whether serum bilirubin levels were correlated with disease severity in patients with pneumoconiosis; the correlation analysis was performed in patients with pneumoconiosis; the results indicated that serum direct bilirubin had a negative correlation with radiological severity in patients with pneumoconiosis (r � −0.320; p � 0.032); no correlation between serum indirect bilirubin and radiological severity was observed in patients with pneumoconiosis.

Multiple Linear-Regression Analysis with Serum Direct
Bilirubin as Dependent Variable. Multiple linear-regression analysis was used to identify independent determinants associated with serum direct bilirubin in patients with pneumoconiosis. When sex, age, smoking history, alanine aminotransferase, aspartate aminotransferase, and radiological severity were included as independent variables, the multiple linear-regression analysis revealed that serum direct bilirubin levels were independently associated with radiological severity in patients with pneumoconiosis (beta = −0.459; p = 0.005), as shown in Table 2.

Discussion
Over the years, serum bilirubin has been reported to be associated with various diseases. For instance, lower serum bilirubin is associated with disease activity in patients with Crohn's disease [16], and lower serum bilirubin levels are  noticed in patients with coronary artery disease [17]. Recently, serum bilirubin has been introduced as a potential biomarker in patients with pneumoconiosis [18]. In our study, serum direct bilirubin levels were lower in patients with pneumoconiosis with stage III than in those with I/II stage, and an association between serum direct bilirubin and radiological severity was observed in patients with pneumoconiosis.
Bilirubin as an endogenous lipid-soluble antioxidant has been highlighted its antioxidant efects [19]. Indeed, serum bilirubin may be a useful parameter to refect oxidative stress in vivo [20], and bilirubin has much stronger antioxidant activity than many other antioxidants [21]; there is a correlation between plasma bilirubin and oxidative-stress markers in HIV-infected patients [22]. In addition to the efects on the oxidative stress, bilirubin also plays a role in anti-infammation and contributes to improve tissue injury through its anti-infammatory mechanism in sepsis [23]; serum bilirubin is correlated with low serum C-reactive protein levels in apparently healthy adults [24]. Tere is an inverse correlation between serum bilirubin and high sensitive C-reactive protein in patients with metabolic syndrome, lower serum bilirubin levels are involved in enhanced low-grade systemic infammation [25].
Moreover, the signifcant inverse correlation between serum bilirubin and infammatory markers has also been suggested in patients with coronary artery disease [26]. Importantly, bilirubin is helpful for inhibiting lung infammation due to its excellent antioxidant and antiinfammatory properties [9,10]. Tus, given that bilirubin may be consumed due to prolonged infammation and oxidative stress in pneumoconiosis; lower serum bilirubin levels may be caused due to an overconsumption of bilirubin in patients with pneumoconiosis.
Interestingly, no signifcant correlation between serum indirect bilirubin and radiological severity was observed among patients with pneumoconiosis. In fact, indirect bilirubin is insoluble in water, and it is tightly bound to albumin [27]; direct bilirubin is weakly bound to albumin and is easily separated from albumin and translates into active form compared with the other bilirubin subtypes when direct bilirubin acts on a molecule or organ to perform its function [28]. Tus, direct bilirubin may carry out its functions of anti-infammation and antioxidative stress in patients with pneumoconiosis.
Recently, a relationship between serum bilirubin and lung function decline has been demonstrated in patients with chronic obstructive pulmonary disease [29]. Apperley S    [30] suggested that serum bilirubin is associated with forced expiratory volume in 1 s in patients with mild chronic obstructive pulmonary disease. Tese fndings suggest that lung function may infuence serum bilirubin levels in chronic obstructive pulmonary disease. In our study, to avoid an infuence from the decline in lung function on the association between serum bilirubin and radiological severity in patients with pneumoconiosis, our study design excluded chronic obstructive pulmonary disease in patients with pneumoconiosis. In addition, smoking is a signifcant contributor for reduced lung function [31], so we further adjusted smoking in the multiple linear-regression analysis, thereby avoiding the efects of smoking on present results in patients with pneumoconiosis.
Regarding the limitations of the study. First, the sample size of the current study is small, especially for subgroup analysis of pneumoconiosis patients. Second, our study did not clarify the efects of anti-infammatory or antioxidant therapy such as glucocorticoid use on serum bilirubin in patients with pneumoconiosis, particularly for inhaled therapy of glucocorticoids. Tird, our study did not analyze the association between bilirubin and infammation or oxidative-stress marker in patients with pneumoconiosis.

Conclusions
In conclusion, our study demonstrates a negative association between serum direct bilirubin and disease severity in patients with pneumoconiosis. However, further studies are warranted to validate the role of serum bilirubin in patients with pneumoconiosis.

Data Availability
Te data used to support the fndings of this study can be obtained from the corresponding author upon reasonable request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.