Serum bilirubin and the risk of chronic obstructive pulmonary disease exacerbations
Brown et al. Respiratory Research
Serum bilirubin and the risk of chronic obstructive pulmonary disease exacerbations
Kirstin E. Brown 0 1 3
Don D. Sin 2 3
Helen Voelker 0 3
John E. Connett 0 3
Dennis E. Niewoehner 0 1 3
Ken M. Kunisaki 0 1 3
for the COPD Clinical Research Network 3
0 University of Minnesota , Minneapolis, MN , USA
1 Minneapolis VA Health Care System , Pulmonary, Critical Care, and Sleep Apnea (111N), One Veterans Drive, Minneapolis, MN 55417 , USA
2 University of British Columbia , Vancouver, BC , Canada
3 Funding Study supported by National Heart, Lung, and Blood Institute awards to COPD Clinical Research Network sites: U10 HL074407, U10 HL074408, U10 HL074409, U10 HL074416, U10 HL074418, U10 HL074422, U10 HL074424, U10 HL074428, U10 HL074431, U10 HL074439, U10 HL074441. Study also supported by Canadian Institutes for Health Research
Background: Bilirubin is a potent anti-oxidant and higher serum concentrations of bilirubin have been associated with better lung function, slower lung function decline, and lower incidence of chronic obstructive pulmonary disease (COPD). We sought to determine whether elevated bilirubin blood concentrations are associated with lower risk for acute exacerbations of COPD (AECOPD). Methods: We performed a secondary analyses of data in the Simvastatin for Prevention of Exacerbations in Moderate-to-Severe COPD (STATCOPE) and the Azithromycin for Prevention of Exacerbations of COPD (MACRO) studies. We used time-dependent multivariable Cox proportional hazards analyses, using bilirubin concentrations prior to first AECOPD as the exposure variable and time to first AECOPD as the outcome variable. STATCOPE was used for model development, with validation in MACRO. Results: In STATCOPE (n = 853), higher bilirubin was associated with a lower but statistically insignificant hazard for AECOPD, (adjusted hazard ratio [aHR] 0.89 per log10 increase [95%CI: 0.74 to 1.09; p = 0.26]). In the validation MACRO study (n = 1018), higher bilirubin was associated with a significantly lower hazard for AECOPD (aHR 0.80 per log10 increase [95%CI: 0.67 to 0.94; p = 0.008]). Conclusions: Bilirubin may be a biomarker of AECOPD risk and may be a novel therapeutic target to reduce AECOPD risk. Trial registrations: ClinicalTrials.gov NCT01061671 (registered 02 February 2010) and ClinicalTrials.gov NCT00325897 (registered 12 May 2006).
Bilirubin; Biomarker; Pulmonary disease; Chronic obstructive
Acute exacerbations of chronic obstructive pulmonary
disease (AECOPD) are associated with accelerated lung
function decline, lower quality of life, increased mortality,
and higher healthcare costs [
pathogenesis is complex, but oxidative stress is commonly
observed in AECOPD.
Anti-oxidant interventions such as carbocysteine and
N-acetylcysteine have been shown to reduce risk for
AECOPD in some [
] but not all  trials. These
interventions are targeted at increasing intracellular and
extracellular concentrations of glutathione, a major
endogenous antioxidant. Bilirubin is another potent
antioxidant that protects lipids against oxidant stress and inhibits
membrane-bound nicotinamide adenine dinucleotide
phosphate oxidase, which is a large intracellular source of
reactive oxygen species [
These antioxidant mechanisms may help explain why
several large observational studies have shown that
higher serum bilirubin concentrations are associated
with better lung function [
], slower rate of FEV1
decline over 3 to 9 years [
] and a lower incidence of
COPD, lung cancer, and all-cause mortality [
Together, these data suggest that higher serum bilirubin
concentrations are associated with a lower risk of
incident COPD and lower rates of COPD disease
progression. No studies have investigated the association
between serum bilirubin concentrations and AECOPD.
Given the important role of oxidant stress in AECOPD
pathogenesis, we hypothesized that higher serum
concentrations of bilirubin would be associated with a
lower risk of AECOPD. We tested this hypothesis using
data from two large multi-center cohorts of patients
with COPD at high risk of AECOPD.
We performed secondary analyses of data in the
Simvastatin for the Prevention of Exacerbations in
Moderate-to-Severe COPD (STATCOPE;
ClinicalTrials.gov NCT01061671) and the Macrolide
Azithromycin to Prevent Rapid Worsening of Symptoms
Associated With Chronic Obstructive Pulmonary Disease
(MACRO; ClinicalTrials.gov NCT00325897) studies.
STATCOPE and MACRO were designed as multi-center
randomized controlled trials to test the efficacy of daily
simvastatin (STATCOPE) and daily azithromycin
(MACRO) to reduce the risk of acute exacerbations of
COPD (AECOPD). The COPD Clinical Research
Network conducted both studies with funding from the
US National Heart, Lung and Blood Institute and
Canadian Institutes of Health Research (for STATCOPE).
The studies were approved by each participating center’s
institutional review board and all study participants
provided informed consent for study participation.
The design and results of both studies have been
]. Inclusion criteria in both studies
included age ≥ 40 years, post-bronchodilator forced
expiratory volume in one second (FEV1)/forced vital
capacity (FVC) <70%, FEV1 < 80% of predicted, ≥10
pack-year smoking history, and an increased risk of
AECOPD (defined as home oxygen use, systemic
corticosteroid or antibiotic usage for AECOPD, or having
an emergency department visit or hospitalization for
AECOPD in the year prior to study entry). Exclusion
criteria included alcoholism and active liver disease
(defined as transaminase elevations >1.5 times the upper
limit of normal in STATCOPE, and >3 times the upper
limit of normal in MACRO). STATCOPE additionally
excluded those already treated with statins, those with
indications to be on a statin according to the Adult
Treatment Panel III risk stratification, and those with
contraindications to statins. MACRO additionally
excluded those with asthma, a resting heart rate greater
than 100 beats per minute, a prolonged corrected QT
(QTc) interval (>450 msec), the use of QT-prolonging
medications, or hearing impairment.
STATCOPE was performed at 45 sites, and study
follow-up time ranged from 21 to 1263 days, with a
median (interquartile range) of 635 (329 to 990) days.
The wide range of follow-up time in STATCOPE was
largely due to the recommendation by the Data Safety
and Monitoring Board for early termination of the trial,
at a time when participants were still being actively
recruited into the study. In contrast, MACRO continued
to its planned closure date and had a follow-up time that
ranged from 0 to 380 days, with a median of 200 (IQR
60 to 357) days. MACRO was performed at 17 sites.
AECOPD was defined identically in both studies as a
complex of respiratory symptoms (increased or new
onset) of more than one of the following: cough, sputum,
wheezing, dyspnea, or chest tightness with a duration of at
least three days requiring treatment with antibiotics or
systemic steroids. Study personnel assessed AECOPD
status monthly via clinic visits or telephone contacts.
Bilirubin concentrations were measured at baseline in
both studies. In STATCOPE follow-up bilirubin was
measured at months 6, 12, 18, and 24. In MACRO,
follow-up bilirubin was measured more frequently, at
months 1, 3, 6, 9, and 12.
We first utilized STATCOPE to develop and calibrate our
analytic model and then validated the model using data
from MACRO. The rationale for this ordering was due to
several limitations of the STATCOPE cohort, compared to
the MACRO cohort for this analysis, including
STATCOPE’s smaller sample size, variable follow-up times, and
less frequent bilirubin measurements (every 6 months in
STATCOPE vs. every 1–3 months in MACRO).
We included all study participants who had at least one
bilirubin measurement and at least one follow-up contact.
One hundred five participants were in both STATCOPE
and MACRO; these participants were included in the
STATCOPE development/calibration dataset and were
then excluded from the MACRO validation analysis.
We used time-dependent multivariable Cox
proportional hazards analyses, using log10-transformed bilirubin
concentrations prior to first AECOPD as the exposure
variable of interest and time to first AECOPD as the
outcome variable of interest. In contrast to traditional (i.e.
non-time dependent) Cox models using only a static
measure of exposure, time-dependent models account for
intrinsically variable exposures that might influence
outcomes (in our case serum bilirubin that has been
shown to vary between several visits over a year [
Time-dependent models also can be more robust than
static models, as they utilize all available data. Due to the
availability of repeated bilirubin measurements in
both of our cohorts, we elected to use this
Our development and calibration model included
analyses of several clinical variables treated as continuous
or categorical data, along with interaction terms.
Covariates were those previously shown to affect AECOPD risk
including age, sex, race, body mass index (BMI), chronic
bronchitis, respiratory health status (as assessed by the St.
George’s Respiratory Questionnaire [SGRQ] score), ethanol
consumption, smoking status, post-bronchodilator
FEV1%predicted, inhaled medications, use of supplemental
oxygen, hospitalization or emergency department visit
within the previous year, and steroid or antibiotic use
within the previous year. Although simvastatin was shown
to not affect AECOPD risk, we felt it important to also
include treatment assignment (i.e. simvastatin vs. placebo)
into the initial full development model. Covariates from this
full STATCOPE development model were included in a
final reduced STATCOPE model if they were significant at
p < 0.10 by backwards stepwise regression; we forced
treatment assignment into the reduced model also. We did not
access the MACRO dataset until after the final reduced
STATCOPE model was agreed upon.
In the better-powered MACRO validation analysis, we
applied the final STATCOPE model, and we forced
treatment assignment (i.e. azithromycin vs. placebo) into
the model, due to azithromycin’s proven effect in
reducing AECOPD risk and unclear effects on bilirubin
Statistical analyses were conducted using SAS 9.3
(SAS Institute, Cary, NC, USA).
Among the 885 participants enrolled in STATCOPE,
853 had bilirubin concentrations measured and had at
least one follow-up contact for determination of
AECOPD status. These 853 participants formed the
development/calibration sample for this study. Mean age
of these STATCOPE participants was 62 years, 56% were
male, 30% were current smokers, mean FEV1 was 1.19 L
(42% of predicted), and 51% had been to an emergency
department or hospitalized for AECOPD within the year
prior to enrollment. Mean bilirubin concentrations were
between 0.62–0.65 mg/dL, which is well within the
general population normal range of 0.20–1.20 mg/dL.
Additional baseline characteristics of these STATCOPE
participants are presented in Table 1.
Of these STATCOPE participants, 475 (56%)
experienced an AECOPD during the study. Covariates that
were independently associated (at p < 0.10) with time to
first AECOPD included male sex, black race, BMI,
chronic bronchitis, supplemental oxygen use, St.
Abbreviations: AECOPD acute exacerbation of chronic obstructive
pulmonary disease, BMI body mass index, ED emergency department,
FEV1 forced expiratory volume in one second, FVC forced vital
capacity, GOLD Global Initiative for Chronic Obstructive Lung Disease
George’s Respiratory Questionnaire (SGRQ) score,
inhaler use, and steroid or antibiotic use within the past
one year (Table 2). Covariates that were not
independently associated with time to AECOPD included age,
hospitalization for AECOPD within the previous year,
ethanol use, FEV1% predicted, and current smoking.
Follow-up bilirubin concentrations were not different
between those assigned to simvastatin vs. placebo.
Male sex - no. (%)
Race - no. (%)
Mean Serum Bilirubin (mg/dL)
Alcohol consumption, drinks per day
0.48 ± 0.85 0.40 ± 1.03
Currently smoking - no. (%)
Oxygen use - no. (%)
ED visit or hospitalized for AECOPD in year
prior to enrollment - no. (%)
FEV1/FVC ratio, %
Post-bronchodilator FEV1, L
Post-bronchodilator FEV1, % predicted
GOLD category - no. (%)
Steroids or antibiotics in previous year - no. (%) 715 (84%) 865 (85%)
(n = 853)
In the STATCOPE development dataset, higher bilirubin
concentrations were independently associated with a lower
but statistically insignificant hazard ratio for AECOPD
(adjusted hazard ratio [aHR] in the final reduced model of
0.89 per log10 increase in bilirubin [95%CI: 0.74 to 1.09;
p = 0.26) (Table 3).
This final model was then applied to the larger,
betterpowered MACRO validation cohort. Of the 1142
MACRO participants, 1024 did not participate in
STATCOPE. 1018 of the 1024 participants had a measured
bilirubin concentration with at least one follow-up
contact for AECOPD status determination and this
comprised the validation cohort. MACRO participants were
similar to STATCOPE participants, with a mean age of
66 years, 60% were male, 22% were current smokers,
and mean FEV1 was 1.12 L (40% of predicted) (Table 1).
640 (63%) of MACRO participants experienced an
AECOPD during the study. Follow-up bilirubin
concentrations were not different between those assigned to
azithromycin vs. placebo. In this validation dataset,
higher bilirubin concentrations were independently
associated with a statistically significantly lower hazard ratio
for AECOPD. The point estimate was similar to that
observed in STATCOPE, but with a narrower confidence
interval (aHR 0.80 per log10 increase in bilirubin [95%CI:
0.67 to 0.94; p = 0.008]) (Table 4).
Our data lend support to the hypothesis that higher
circulating bilirubin concentrations are associated with a
lower risk of AECOPD. Our findings are consistent with
other observational data suggesting clinically important
cardiopulmonary health benefits associated with higher
Several studies in the cardiac literature have
demonstrated that higher blood bilirubin concentrations are
associated with a lower risk of cardiovascular disease
including peripheral vascular disease, carotid
intimalmedial thickness, and stroke [
]. A study in the
Framingham Heart Study Offspring cohort showed that
among 1780 individuals with 24 years of follow-up, those
with higher bilirubin due to a genetic polymorphism
affecting the UGT1A1 enzyme of bilirubin metabolism
(the enzyme defect that leads to Gilbert’s syndrome) had
approximately one-third the risk of cardiovascular events
compared to wild-type carriers with normal bilirubin
]. The significant relationship between
genetically determined bilirubin and cardiovascular
events (an example of a so-called ‘Mendellian
randomization’ study design) provides some additional
support to a causal relationship. However, a more recent
meta-analysis did not support an association between
genetically elevated bilirubin and reduced risk of
ischemic heart disease [
], thus, the potential protective role
of bilirubin in cardiovascular disease pathogenesis is not
In addition to the data suggesting a potential cardiac
benefit to higher blood bilirubin concentrations,
emerging data suggest pulmonary benefits as well. A
cross-sectional, population-based spirometry study of
4195 smokers and non-smokers in Switzerland showed
that elevated serum concentrations of bilirubin and a
genetic polymorphism associated with higher bilirubin
concentrations were both independently associated with
better lung function [
These cross-sectional findings were supported by a
subsequent longitudinal study of 4680 North American
smokers aged 35 to 60 years old with mild to moderate
COPD at study entry, where higher serum bilirubin at
baseline was associated with higher baseline FEV1 and a
significantly slower rate of FEV1 decline over 3 to 9 years
of prospective follow-up [
]. In this cohort of persons
with COPD, there was also an association between
higher serum bilirubin and a lower risk of coronary
heart disease mortality over 15 years.
The largest study to investigate the association
between serum bilirubin and pulmonary outcomes was
conducted using a UK primary care research database
]. In this longitudinal sample of 504,206 adults with a
median follow-up time of 8 years, higher serum bilirubin
concentrations were associated with a significantly lower
incidence of COPD (adjusted incidence rate ratio of 0.92
[0.89 to 0.95] per 0.1 mg/dL increase in bilirubin in men
and 0.89 [0.86 to 0.93] in women]. Higher bilirubin was
also associated with a lower incidence of lung cancer
and all-cause mortality.
Together, these data suggest that higher bilirubin
concentrations are associated with a lower risk of
incident COPD and lower rates of disease progression.
We extend these data by demonstrating that elevated
serum bilirubin concentrations are associated with a
lower risk of AECOPD, one of the most important
clinical outcomes for patients with COPD.
Our study has several strengths, including the ability
to perform analyses in two large, multi-center trials that
used very similar entry criteria and carefully collected
AECOPD data, the primary outcome of both original
studies. Unlike many epidemiologic studies that conduct
analyses within only a single cohort, we were uniquely
able to develop our analytic model in an exploratory
fashion in the STATCOPE dataset, knowing from the
outset that this would be a somewhat underpowered
cohort due to infrequent bilirubin measurements,
smaller sample size, fewer exacerbations, and variable
follow-up time. We were thus able to reserve the
betterpowered MACRO dataset for validation purposes only.
Moreover, the serial measurements of serum bilirubin
during the observational period, especially the frequent
measures in the MACRO validation dataset, enabled us
to use a dynamic and flexible time-dependent Cox
model to determine the relationship between bilirubin
and incident exacerbations.
Our study has some limitations, including its reliance
on bilirubin assays performed in clinical laboratories at
study sites, rather than in a central laboratory. However,
bilirubin is a well-established assay with well-established
standard operating procedures in clinical laboratories,
and an inter-laboratory coefficient of variation of only
1% to 3% [
]. The study population was also limited to
those from North America at high risk of AECOPD, so
our data do not apply to those at lower risk of COPD
and may not apply to patients with COPD in other
regions of the world. Perhaps most importantly, our study
is observational and therefore unable to prove causality.
High bilirubin concentrations could potentially be
confounded by other healthy behaviors associated with these
Our study was not designed to answer mechanistic
questions regarding how bilirubin might confer
pulmonary benefits, but biologic plausibility comes from
publications demonstrating that bilirubin is a potent
]. Indeed, bilirubin may be the most potent
in vivo antioxidant to protect lipids against oxidant
stress, tissue degeneration and death [
]. Smoking and
other environmental oxidant insults significantly reduce
serum bilirubin concentrations, but shortly after
smoking cessation, serum bilirubin rapidly increases [
recent study using a rat model of COPD showed that
exogenous administration of bilirubin (as a therapeutic
agent) reduced lung and systemic inflammation,
suppressed regional oxidative lipid damage and prevented
progression of histological changes of emphysema [
Bilirubin also inhibits membrane-bound nicotinamide
adenine dinucleotide phosphate oxidase, which is a large
intracellular reactive oxygen species source [
In summary, cellular and animal model data suggest
that higher concentrations of bilirubin provide
antioxidant benefits to the lung, while observational human
data support the notion that higher bilirubin
concentrations are associated with better COPD-related outcomes.
Our data are consistent with these observations and
suggest that higher bilirubin concentrations are associated
with a lower risk for AECOPD.
Among individuals with moderate-to-severe COPD
without active liver disease, higher serum bilirubin
concentrations are independently associated with a lower
risk for AECOPD. Bilirubin may be a novel biomarker of
AECOPD risk and may represent a novel therapeutic
target for future investigations.
We thank the participants who participated in the STATCOPE and MACRO
The views expressed in this article are those of the authors and do not
necessarily represent the views of the Minneapolis VA Health Care System,
the U.S. Department of Veterans Affairs, the National Institutes of Health, the
U.S. Government, or the authors’ affiliated academic institutions.
The funders had no role in the conduct, analysis, writing, or decision to
submit for publication, for either the original studies or this current analysis.
Availability of data and materials
The datasets analyzed during the current study are available from the
corresponding author on reasonable request.
KEB made substantial contributions to the conception of the work and
interpretation of data, drafted the manuscript, revised it critically for important
intellectual content, and approved the final version to be published. DDS made
substantial contributions to the conception of the work and interpretation of
data, revised the manuscript critically for important intellectual content, and
approved the final version to be published. HV made substantial contributions to
the acquisition, analysis, and interpretation of data, and approved the final
version to be published. JEC made substantial contributions to the conception
of the work, the acquisition, analysis, and interpretation of data, revised the
manuscript critically for important intellectual content, and approved the final
version to be published. DEN made substantial contributions to the analysis and
interpretation of data, revised the manuscript critically for important intellectual
content, and approved the final version to be published. KMK made substantial
contributions to the conception and design of the work, the acquisition, analysis,
and interpretation of data, drafted the manuscript and revised it critically for
important intellectual content, and approved the final version to be published.
Ethics approval and consent to participate
Ethics approval was obtained at each study site in the original trials from
which this secondary analysis was conducted. All participants provided
Consent for publication
DDS has received advisory board honoraria, research funding and speaking
fees from AstraZeneca, meeting honoraria and research funding from
Boehringer Ingelheim, research funding from Merck Frosst, and advisory
board honoraria from Novartis.
DEN has received consulting fees from GlaxoSmithKline, Boehringer
Ingelheim, and AstraZeneca.
KEB, HV, JEC, and KMK declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in
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