Serum bilirubin and chronic obstructive pulmonary disease (COPD): a systematic review

Background Bilirubin is a potent antioxidant and higher serum bilirubin levels have been associated with improved COPD outcomes. We performed a systematic review to evaluate the association between serum bilirubin levels and lung function (FEV1), prevalence/incidence of COPD, acute exacerbations of COPD, respiratory health status, and mortality. Methods MEDLINE® and Embase were searched using Ovid® (search updated October 1st, 2019). We included studies that measured serum bilirubin levels and outcomes of interest in adults with or without underlying lung disease. We excluded studies of those with liver disease or drug-induced elevations in bilirubin. We used the Newcastle–Ottawa scale to assess individual study risk of bias (ROB) and the US Agency for Healthcare Research and Quality—Evidence Based Practice tool to assess overall strength of evidence (SOE). Two authors independently determined eligibility, performed data abstraction, assessed ROB, and determined SOE. Results Thirteen studies (5 low risk of bias, 3 moderate and 5 high risk) were included. We found low strength of evidence for the association between higher bilirubin levels and lower risk of acute exacerbations of COPD (2 studies), mortality (3 studies), COPD diagnosis (4 studies), and lung function (FEV1) (8 studies). We found insufficient evidence on the relationship between serum bilirubin and respiratory health status/exercise capacity (1 study) and airflow obstruction (FEV1/FVC ratio) (4 studies). Conclusion Higher bilirubin levels may be associated with lower mortality and improved COPD outcomes. Randomized trials are needed to evaluate the effect of medications that raise serum bilirubin on COPD outcomes. PROSPERO registration: CRD42019145747.


Background
Chronic obstructive pulmonary disease (COPD) is among the leading causes of death worldwide [1]. Although the mortality rate for COPD has been decreasing in some countries, it has continued to rise in others [2]. In addition to mortality, COPD is a leading cause of respiratory-associated disability, health care utilization, and economic burden, with much of the cost and morbidity driven by acute exacerbations [3,4].
Bilirubin, the main metabolic end-product of heme degradation, is a potent anti-oxidant that scavenges peroxyl radicals and inhibits membrane-bound nicotinamide adenine dinucleotide phosphate oxidase (NADPH) oxidase, which is a large intracellular source of reactive oxygen species [5][6][7][8][9]. Oxidative stress is increased in stable COPD, with further increases during acute exacerbations of COPD (AECOPD) [10]. NADPH oxidase plays a role in lung signaling and deleterious remodeling, and has been implicated in a variety of lung diseases, including COPD [11,12]. Observational studies have previously suggested that higher serum bilirubin concentrations, but still within the normal range of serum bilirubin (what we have termed 'benign elevations'), are associated with decreased COPD diagnosis, lower risk of AECOPD, slower rate of lung function decline, and lower all-cause mortality [13][14][15][16][17][18][19]. However, other studies have found no effect of serum bilirubin on similar outcomes [20][21][22]. The pathogenesis of COPD is complex, but oxidative stress is commonly observed in AECOPD and has been associated with faster decline in the forced expiratory volume in 1 s (FEV 1 ) [12]. Therefore, the potent antioxidant effect of bilirubin could be a novel therapeutic intervention [9].
We evaluated the evidence on potential protective benefits of benign elevations in serum bilirubin in COPD.
Our key questions (KQ) were: KQ1 Is serum bilirubin associated with better lung function as measured by FEV 1 ? KQ2 Is serum bilirubin associated with lower prevalence/incidence of COPD and/or better (higher) FEV 1 /FVC ratio? KQ3 Is serum bilirubin level associated with improved clinically relevant outcomes (respiratory events/exacerbations, respiratory symptoms, respiratory health status, and mortality)?

Methods
This systematic review was conducted according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) standards [23]. Our protocol was registered in PROSPERO (registration number CRD42019145747).

Data sources and searches
We searched MEDLINE ® and Embase databases using Ovid ® (search updated October 1st, 2019) for peerreviewed, English language studies. The search strategy for the MEDLINE ® search is provided in Additional file 1: Appendix 1.

Study selection
Two authors (AKB and DMM) independently reviewed each study at the abstract and full-text level to determine eligibility. Disagreements were resolved through discussion and evaluation by a third reviewer, if needed.
Reports were eligible if they were randomized or observational (cohort, cross-sectional, case control) studies that enrolled participants greater than 18 years of age. We did not exclude studies based on pulmonary function or pulmonary diagnoses and included studies conducted in both general populations and in samples restricted to those with obstructive lung disease. We excluded case reports and case series, studies published in only abstract form, and those without a full text publication in English. We did not have a numerical cutoff for bilirubin levels because there are no defined criteria for what constitutes a pathologic elevation in bilirubin, but we excluded studies evaluating elevations in serum bilirubin in the context of hepatobiliary diseases and drug-induced liver injury. Eligible studies reported serum bilirubin levels and any outcome of interest related to our three key questions.

Outcome measures
We examined clinically important outcomes by cross sectional measures and longitudinal measures. Clinical effect sizes are included in Additional file 1: Appendix 2.

Data extraction and quality assessment
Data extraction was independently completed by two reviewers (DMM and AKB). We extracted data on study characteristics (first author, year published, sample size, and study population), inclusion criteria, serum bilirubin levels, outcome measurements, methodology of outcome assessments, and covariates included in modeling the outcome of interest. If a study included both cross-sectional and longitudinal analyses, the longitudinal analysis results were preferentially selected for this systematic review.
We assessed risk of bias for observational studies using the Newcastle-Ottawa Scale (NOS), a validated tool used to assess quality of nonrandomized studies [24,25]. The NOS uses a star system to assess a study on three main domains: selection of study groups (four star scale), comparability of study groups (two star scale), and ascertainment of either exposure or outcome of interest (three star scale). There are no accepted criteria for assigning risk of bias based upon the number of stars. We a priori defined studies as "low" risk of bias if they met all nine stars across the assessment criteria. "Moderate" risk of bias was assigned to studies that met seven or eight stars. "High" risk of bias was assigned to studies that met six or fewer stars, if the reviewers could not ascertain star criteria for a study, and for studies with n ≤ 200. The Newcastle-Ottawa Scale is shown in Additional file 1: Appendix 3.

Data analysis
Data synthesis for this review was limited to qualitative summary due to high clinical heterogeneity (variation in study population and outcomes measured) and methodological heterogeneity (variation in study design, exposure measures, outcome measures, and model covariates). We were unable to perform quantitative analysis of FEV 1 (mL/year) because the 95% confidence interval (CI) was only available for one study.

Overall strength of evidence
Two reviewers independently assessed the strength of evidence based on methods guidance provided by the Evidence Based Practice Center (EPC) established by the U.S. Agency for Healthcare Research and Quality (AHRQ), adapted for exposure (serum bilirubin level) rather than an intervention [26]. A complete description of the assessment of strength of evidence is included in Additional file 1: Appendix 2.
Studies excluded after full-text review are listed in Additional file 1: Appendix 4.
We found an inverse association of bilirubin levels with all-cause mortality ("low" strength of evidence). Though the largest study showed a statistically significant association between higher serum bilirubin and lower mortality [17], the overall body of evidence lacked precision and consistency.

Acute exacerbations of COPD
Two studies evaluated the association between serum bilirubin and AECOPD [19,27]. Both found higher serum bilirubin was associated with significantly lower rates of AECOPD [19,27]. One low ROB study was a secondary analysis of two randomized trials of participants with COPD enriched for participants prone to exacerbations. The model was developed in one cohort (n = 853), where bilirubin was not a significant predictor of COPD exacerbation (adjusted hazard ratio per log 10 increase in bilirubin = 0.89; 95% CI 0.74-1.09, p = 0.26) but in the validation cohort (n = 1018) the relationship between higher serum bilirubin and time to first AECOPD was significant (adjusted hazard ratio per log 10 increase in bilirubin = 0.80; 95% CI 0.67-0.94, p = 0.008) [19]. The other study (n = 535), which had moderate risk of bias, Fig. 2 Summary of measured outcomes. Each row represents an outcome of interest. Columns are divided by statistical significance, with the no difference column representing studies that found no statistically significant relationship for the outcome of interest. Individual studies are represented by circles and are colored green for low risk of bias, yellow for moderate risk of bias, and red for high risk of bias. The size of each circle is representative of the number of participants in the study, but not directly proportional. Each study can be represented by more than one circle if it investigated more than one outcome of interest was in a cohort of patients with obstructive lung disease and found that higher serum bilirubin was associated with lower rates of AECOPD [estimated mean from regression 0.62 (standard error 0.18; p = 0.001)] [27].
We found an inverse association between bilirubin levels and AECOPD ("low" strength of evidence). Though both studies found a statistically significant association between higher serum bilirubin and lower rates of AECOPD, one study found a statistically significant relationship only in the validation cohort, not the development cohort and the other had a moderate risk of bias [19,27]. There were no data specific to the association between serum bilirubin and risk of hospitalization for AECOPD.

Respiratory health status and exercise capacity
One moderate ROB study evaluated respiratory health status (CAT and SGRQ) and exercise capacity (6-min walk test) and found no significant association between serum bilirubin and any of these measures [27]. We determined that there was insufficient evidence on the association of bilirubin with respiratory health status and exercise capacity.

COPD diagnosis
Four studies evaluated the association between serum bilirubin and COPD diagnosis [15,17,20,27]. One large (n = 504,206) longitudinal cohort study with low risk of bias found a small but statistically significant decrease in  [17].
Three studies were small, cross-sectional, and had high risk of bias; none found a significant relationship [22,28,29]. We found an inverse association between bilirubin levels and COPD diagnosis. We assigned "low strength evidence" based upon the moderate overall bias, inconsistent effects, and imprecision of reported outcomes.

Lung function (FEV 1 )
Results for studies evaluating the association between serum bilirubin and FEV 1 are shown in Table 2. Among the three studies (all low ROB) [13,15,20] that evaluated longitudinal change in FEV 1 , two found a statistically significant relationship. One was a general population cohort with a mean follow-up of 5.4 years (FEV 1 declined 13.09 mL/yr slower for every 1 unit increase in natural log of bilirubin) and the other was a longitudinal cohort of smokers with a mean FEV 1 /FVC ratio of < 0.7 with up to 9 years of follow-up (FEV 1 declined 57.0 mL/yr in the highest quintile of bilirubin and 66.3 mL/year in the lowest) [13,15]. The study that did not find a statistically significant relationship was from a smaller cohort of young, HIV positive adults who were followed for a median of 3.9 years [20].
Five studies [14,16,18,21,27], 3 moderate ROB [14,18,27] and 2 high ROB [16,21] evaluated cross-sectional associations between bilirubin and FEV 1. Two studies found a statistically significant relationship between higher FEV 1 and higher serum bilirubin [14,16]. One low risk of bias cohort sample from England, Wales, and Scotland [14] evaluated 5,362 people born in the same week in 1946 and the other high risk of bias study [21] was from a nationally representative cohort in the United States. Three found no significant relationship between FEV 1 and serum bilirubin levels [18,21,27].
We found an association between higher bilirubin levels and improved lung function (FEV 1 ). We assigned a "low" strength of evidence due to the moderate overall risk of bias of the eight studies evaluated, clinically small effect, inconsistent effects, and imprecision of the outcome (unreported or wide confidence intervals).

Airflow obstruction (FEV 1 /FVC ratio)
Four studies evaluated the association between serum bilirubin and FEV 1 /FVC ratio, a spirometric marker of obstructive lung disease [15,18,20,27] (Table 3). Three of the studies were longitudinal [15,20,27]. One longitudinal study with low risk of bias found an association between higher baseline bilirubin levels and faster decline in FEV 1 /FVC ratio [ß for % decline in FEV 1 / FVC per 1 unit change in natural log of bilirubin (mg/ dL) = 1.69; p < 0.001] [15]; two found no significant  [20,27]. A cross-sectional population-based analysis with moderate risk of bias found a significant association between higher bilirubin levels and less airflow obstruction [ß for % decline in FEV 1 /FVC per 1 unit increase in natural log in bilirubin (μmol/L) 0.5; 95% CI 0.1-1.0, p = 0.012] [18]. There was insufficient evidence to evaluate the relationship between airflow obstruction and serum bilirubin.

Discussion
This report provides the first systematic review of the relationship between "benign" elevations in serum bilirubin levels and COPD clinical outcomes and lung function. Our analysis of data from 13 observational studies found an association between higher serum bilirubin and reduced mortality and improved COPD outcomes and lung function measures (low strength evidence). The effect based on a priori thresholds was deemed small in absolute magnitude. Due to clinical and methodologic heterogeneity and data reporting limitations we were unable to perform a meta-analysis. Instead, we presented a summary of findings across studies and outcomes. The strength of evidence for associations varied by outcome from "insufficient" for quality of life, exercise capacity, and FEV1/FVC ratio to "low" for the relationship between higher serum bilirubin and risk of AECOPD, lung function (FEV 1 ), COPD diagnosis, and mortality. Though we found low strength of evidence, our most consistent finding was the association between serum bilirubin and AECOPD. This is particularly intriguing given the increased oxidative stress observed during and after AECOPD, and the potent anti-oxidant effects of bilirubin [5][6][7][8]12]. Only one of these studies evaluated the correlation between bilirubin and other anti-oxidant markers. They found a statistically significant, but negligible correlation (Pearson's correlation coefficient 0.19, p < 0.05) between bilirubin and the Trolox equivalent antioxidant capacity (TEAC) assay, which has been used as a summary measurement of antioxidant activity [21]. N-acetylcysteine and carbocisteine, which have anti-oxidant and anti-inflammatory properties, may have a role in reduction of AECOPD [30,31]. Though antioxidants have been disappointing when used for the prevention of lung function decline, increased dietary intake of antioxidants may be associated with better lung function outcomes [32,33]. Whether bilirubin levels can be manipulated to provide a similar benefit has not been investigated.
We identified gaps in the existing literature that limited our conclusions. The study designs and populations were heterogenous, preventing quantitative analyses. For example, of the three longitudinal studies analyzing the relationship between serum bilirubin and FEV 1 , one was a cohort of smokers with mild to moderate airflow obstruction from the Lung Health Study, one was a Korean cohort which excluded those with preexisting lung disease, and one was a cohort from a randomized controlled study of young, HIV positive participants [13,15,20]. These studies also used different methods to quantify bilirubin (e.g. quintile, natural log or log 2 transformation). Lastly, one of these studies did not report a 95% confidence interval for the regression coefficient [15]. Similar methodologic and population differences were present for the other outcomes of interest. COPD diagnosis and mortality findings were also influenced mostly by one large study [17]. Though we assigned clinical effect sizes to the outcomes of interest, we were unable to utilize these to assign an overall clinical effect size due to heterogeneity in reporting of outcomes and analysis methods. Lastly, this analysis may be limited by reporting bias, where negative analyses within these studies were not reported, and by the post hoc nature of these studies.
The studies included in this review were all observational, and therefore, we were unable to establish causality. No randomized controlled trials have assessed the effectiveness and harms of increasing bilirubin levels on adults with or at risk for COPD-related morbidity and mortality. Bilirubin excretion is mediated by UDP-glucuronosyl transferase-1A1 (UGT1A1), the enzyme that conjugates bilirubin for excretion into the bile. Polymorphisms in UGT1A1 account for approximately 10-40% of the variation in serum bilirubin levels and these polymorphisms may be associated with improved respiratory function [34][35][36]. Gilbert's syndrome, a genetic cause of benign bilirubin elevations, is caused by a deficiency in UGT1A1. Inhibition of UGT1A1 is a possible target to raise bilirubin levels, and there are already existing medications, such as the HIV antiretroviral atazanavir, that inhibit UGT1A1 and cause reversible elevations in bilirubin [37].
Though not the focus of this systematic review, Horsfall and colleagues examined the relationship between genetic changes underlying Gilbert's syndrome and respiratory outcomes. They found associations with higher FEV 1 and FVC, a relationship which was strongest among heavy smokers who would be at greatest risk of developing COPD [14]. In an analysis of UK Biobank data, Horsfall et al. also found that genetic changes associated with higher bilirubin levels are associated with a decreased risk of lung cancer, a relationship that was also strongest in heavy smokers [36]. The degree of increase in bilirubin and duration of therapy that would be needed to improve COPD and respiratory health outcomes remain unknown, and would need to be investigated in interventional trials. We excluded studies of pathologic elevations in bilirubin as our goal was to analyze whether benign elevations in bilirubin were a possible therapeutic target. Though several of the included studies eliminated participants with bilirubin levels greater than 1.75 mg/ dL for women and 2.34 mg/dL for men [13,17,27] or greater than 0.99 mg/dL for all participants [18], there is no broadly accepted upper limit of normal for bilirubin levels.
This review highlighted areas for further study. The relationship between serum bilirubin levels and patient-centered outcomes such as exercise capacity and respiratory health status should be evaluated in future studies; this has only been done in one study thus far [27]. Randomized controlled trials of interventions that increase serum bilirubin levels should be considered, especially in those prone to AECOPD because they are at greater risk of future events and are in need of additional novel therapies to reduce their AECOPD risk.

Conclusion
Higher serum bilirubin within the normal range may be associated with lower mortality and risk of COPD exacerbations, a reduced incidence in COPD diagnosis and improved lung function Interventional trials are needed to test if medications that raise serum bilirubin levels can safely improve COPD outcomes.